Methods of identifying SENP1 inhibitors

ABSTRACT

Provided herein are methods of detecting binding of an SENP1 polypeptide to a compound and methods for screening for inhibitors of SENP1. Further provided are aqueous compositions comprising SENP1 polypeptides and NMR apparatuses comprising the compositions for NMR analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/247,153, filed Apr. 7, 2014, now U.S. Pat. No. 9,791,447, issued Oct.17, 2017, which claims the benefit of U.S. Provisional PatentApplications 61/809,208, filed Apr. 5, 2013, and 61/813,832, filed Apr.19, 2013, each of which is incorporated herein by reference in itsentirety and for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under NM Grant Nos.R01GM074748, R01GM086171 and R01GM102538. The government has certainrights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 48440-521C01US_ST25.TXT, created onOct. 13, 2017, 22,281 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND

Post-translational modifications with the small ubiquitin-like modifiers(SUMO) are initiated and removed by the activities of SUMO-specificproteases (SENPs). Unlike ubiquitylation, which has one modifier (i.e.,ubiquitin) and one dominant role, namely protein degradation,SUMOylation involves three modifiers (SUMO-1, -2, and -3) and affectsdiverse cellular functions. There are six SENPs, organized into threefamilies based on sequence similarity: SENP1 and 2 that catalyzematuration of SUMO precursors and removal of SUMO-1 and SUMO-2/3conjugates; SENP3 and 5 that preferentially remove SUMO-2/3 conjugates;and SENP6 and 7 that appear to be mainly involved in editingpoly-SUMO-2/3 chains. Recently, another de-SUMOylase has been discoveredthat does not share sequence similarity with the SENPs.

SENP inhibitors with cellular activity would be advantageous forelucidating the role of SUMOylation in cellular regulation and forvalidating SENPs as therapeutic targets. SENP1 and SENP3 are alsopotential targets for developing new therapeutic agents for cancer. Theyregulate the stability of hypoxia-inducible factor 1α (HIF1α), which isa key player in the formation of new blood vessels to support tumorgrowth. SENP1 is also highly expressed in human prostate cancerspecimens and regulates androgen receptor (AR) activities. Androgeninduces rapid and dynamic conjugation of SUMO-1 to AR, while SENP1promotes AR-dependent transcription by cleaving SUMO-1-modified AR.SENP1 overexpression induces transformation of normal prostate glandtissue and facilitates the onset of high-grade prostatic intraepithelialneoplasia. Therefore, at least some members of the SENPs are potentialtargets for developing new cancer therapies.

SUMMARY

Provided herein are methods of detecting binding of an SENP1 polypeptideto a compound and methods for screening for inhibitors of SENP1. Furtherprovided are aqueous compositions comprising SENP1 polypeptides and NMRapparatuses comprising the compositions for NMR analysis.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a picture of a representative Coomassie-stained gel showingcleavage of SUMO-1 and SUMO-2 by SENP1 and SENP2 in the presence ofincreasing concentrations of SPI-01. YSE, fusion SUMO (S) precursorsflanked by YFP (Y) and ECFP (E) at the N- and C-termini, respectively.

FIG. 2 is a picture of representative Coomassie-stained gel showingcleavage of SUMO-1 and SUMO-2 by SENP1 and SENP2 in the presence ofincreasing concentrations of SPI-07. YSE, fusion SUMO (S) precursorsflanked by YFP (Y) and ECFP (E) at the N- and C-termini, respectively.

FIGS. 3A-3C are graphs showing the effects of the panel of inhibitorsshown in Table 1 at inhibiting SENP1, 2 and 7. In 96-well plates, SENPs(50-200 nM) were pre-treated with increasing concentrations of eachcompound, after which DUB-Glo (40 μM final concentration; Promega,Madison, Wis.) was added as substrate. Experiments were performed intriplicate. The amount of cleaved product is proportional to therelative light unit (RLU), which is bioluminescence produced byluciferase catalyzed reaction of luciferin that was produced by SENPcleavage of DUB-Glo.

FIG. 4 is a picture of a gel showing accumulation of SUMO-2/3-modifiedproteins in HeLa cells upon treatment with increasing doses of SPI-01.

FIG. 5 is a picture of a gel showing retention of SUMOylated proteinsduring recovery of HeLa cells from heat shock in the presence of 60 μMSPI-01 and SPI-02.

FIG. 6 is a graph showing superimposition of a section of the 2D¹H-¹⁵N-heteronuclear single quantum coherence (HSQC) spectra of thecatalytically inactive C603S mutant of human SENP1 in the absence (blackcross-peaks) and presence of SPI-01 (grey cross-peaks) at 25° C.Perturbed representative cross-peaks at or near the catalytic site ofSENP1 are labeled.

FIG. 7 is a graph showing the superimposition of a section of the 2D¹H-¹⁵N-HSQC spectra of SUMO-1 precursor showing labeled peaks of theC-terminal residues when free (black) and bound to SENP1-C603S (darkgrey) or both SENP1-C603S and SPI-01 (light grey) at 35° C.

FIG. 8 is a picture showing all SPI-01 perturbed residues on SENP1 (PDBID: 2IY1) labeled and colored in dark grey on the surface representationof SENP1 in complex with SUMO-1 precursor. Perturbed residues that arelocated in the vicinity of the catalytic center of SENP1 or theC-terminus of precursor SUMO-1 are labeled in black and grey,respectively.

FIGS. 9A and 9B are graphs showing enzyme kinetic measurements forSPI-01 indicating a non-competitive mode of inhibition. The data werefit to obtain the indicated kinetic parameters (α, K_(i) and K_(m))using Graphpad Prism. Lineweaver-Burk plot analysis of the data alsoconfirmed non-competitive inhibition.

DETAILED DESCRIPTION

SENP1 is a target for developing new therapeutic agents for cancer. Itregulates the stability of hypoxia-inducible factor 1α (HIF1α), which isa key player in the formation of new blood vesicles to support tumorgrowth. SENP1 is also highly expressed in human prostate cancerspecimens and regulates androgen receptor (AR) activities. SENP1 is alsoa target for developing anti-viral therapeutic agents for infection ofviruses including, but not limited to influenza, cytomegalovirus, herpesvirus, white spot syndrome virus, Epstein-Barr virus, adenovirus andHIV-1, because of the role of SUMOylation in their replication. Asdescribed in the examples below, small molecule inhibitors of SENP1 weresearched for using in-silico screening in conjunction with biochemicalassays. However, the data provided evidence for substrate-assistedinhibitor binding. Thus, using artificial substrates for compoundscreening may be misleading, as the inhibitory effects could besignificantly different from using the physiological substrates.Therefore, embodiments are provided including methods and inhibitors ofSENP1 that confer the non-competitive inhibitory mechanism, as shown bynuclear magnetic resonance (NMR).

For specific SENP proteins described herein (e.g., SENP1), the namedprotein includes any of the protein's naturally occurring forms, orvariants that maintain the protein activity (e.g., within at least 50%,80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to thenative protein). In some embodiments, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring form.In other embodiments, the SENP1 protein is the protein as identified byits NCBI sequence reference. In other embodiments, the SENP1 protein isthe protein as identified by its NCBI sequence reference or functionalfragment thereof.

The term “SENP1” as provided herein includes any of the Sentrin-specificprotease 1 (SENP1) naturally occurring forms, homologs, isoforms orvariants that maintain the protease activity (e.g., within at least 50%,80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to thenative protein). In some embodiments, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring form.In embodiments, the SENP1 protein is the protein as identified by theNCBI sequence reference GI:390131988 or functional fragment thereof. Inembodiments, the SENP1 protein is the protein as identified by theUniProt sequence reference Q9P0U3 or functional fragment thereof. Inembodiments, the SENP1 protein includes the sequence of SEQ ID NO:1, 2,3, 4, 5, 6, or 7. In embodiments, the SENP1 protein is encoded by anucleic acid sequences corresponding to Gene ID: 29843.

As described herein, nuclear magnetic resonance (NMR) approaches haveadvantages over other methods previously employed on SENP1 inidentifying molecules or compounds for further development.Specifically, the methods herein provide for discovery or identificationof compounds or inhibitors that selectively bind SENP1 and not otherSENPs. The methods also provide for identification of compounds orinhibitors that selectively bind SENP1-physiological substrate complexesand not SENP-artificial substrate complexes. Further advantages includesensitivity to binding affinities of a wide range and, thus, allowingfor identification of compounds with physicochemical properties that areamenable for a greater scope for development of leads with superior ADME(absorption, distribution, metabolism, and excretion) attributes.Optionally, the test compounds are Rule-of-three (Ro3) (MW≤300, H-bonddonors/acceptors≤3, c Log P≤3, rotatable bonds≤3) compliant (Congreve etal., Drug Discov. Today 8(19):876-7 (2003); and Erlanson, Top Curr.Chem. 317:1-32 (2011)).

Nuclear magnetic resonance (NMR) studies magnetic nuclei and provideatomic resolution information on the structures of large or smallmolecules and their complexes. The elementary particles, neutrons andprotons, composing an atomic nucleus, have the intrinsic quantummechanical property of spin. The overall spin of the nucleus isdetermined by the spin quantum number I. If the number of both theprotons and neutrons in a given isotope are even, then I=0. In othercases, however, the overall spin is non-zero. A non-zero spin isassociated with a non-zero magnetic moment. It is this magnetic momentthat is exploited in NMR. For example, nuclei that have a spin ofone-half, like Hydrogen nuclei (¹H), a single proton, have two possiblespin states (also referred to as up and down, respectively). Theenergies of these states are the same. Hence the populations of the twostates (i.e. number of atoms in the two states) will be approximatelyequal at thermal equilibrium. If a nucleus is placed in a magneticfield, however, the interaction between the nuclear magnetic moment andthe external magnetic field means the two states no longer have the sameenergy. The NMR frequency (f) is shifted by the shielding effect of thesurrounding electrons. In general, this electronic shielding reduces themagnetic field at the nucleus (which is what determines the NMRfrequency). As a result, the energy gap is reduced, and the frequencyrequired to achieve resonance is also reduced. This shift of the NMRfrequency due to the chemical environment is called the chemical shift,and it explains why NMR is a direct probe of chemical structure. Thechemical shift in absolute terms is defined by the frequency of theresonance expressed with reference to a standard which is defined to beat 0. The scale is made manageable by expressing it in parts per million(ppm) of the standard frequency. Thus, in general, NMR spectral data arereported as chemical shift and are reported in ppm relative to either aninternal standard or other baseline. A more detailed discussion ofnuclear magnetic resonance may be found in, for example, C. P. Slichter,Principles of Magnetic Resonance, 3rd ed., Springer-Verlag, Berlin, pp.1-63 (1990); J. D. Roberts, Nuclear Magnetic Resonance, Mc-Graw-Hill,N.Y., pp. 1-19 (1959); Cohen-Tannoudji et al., Quantum Mechanics, Vol.1, New York, N.Y.: Wiley (1977); WO 2009/027973; WO 2009/029880; WO2009/029896; Hajduk et al., “High-throughput nuclear magneticresonance-based screening,” J. Med. Chem. 42:2315-2317 (1999); andCavanagh et al., Protein NMR Spectroscopy: Principles and PracticeAcademic Press: San Diego (1996), which are incorporated by referenceherein in their entireties.

A variety of NMR approaches have been developed to accelerate NMR dataacquisition (Atreya et al., Methods Enzymol., 394:78-108 (2005)). Forexample, in the field of biological NMR spectroscopy (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press: San Diego (2007)) stableisotope (¹³C/¹⁵N) labeled biological macromolecules are now studied. Theisotope labeling enables one to efficiently record three-dimensional(3D) or four-dimensional (4D)¹³C/¹⁵N-resolved spectra. The most commonlyused biological NMR methods are multi-dimensional andheteronuclear-edited NMR methods. See, for example, Tjandra and Bax,“Direct measurement of distances and angles in biomolecules by NMR in adilute liquid crystalline medium,” Science 1997 278(5340):1111-4 (1997).Erratum in: Science 278(5344):1697 (1997); Clore and Gronenborn, “NMRstructure determination of proteins and protein complexes larger than 20kDa,” Curr Opin Chem Biol. October; 2(5):564-70 (1998); Mittermaier andKay, “Observing biological dynamics at atomic resolution using NMR,”Trends Biochem Sci. 34(12):601-11 (2009); and Wüthrich, Kurt, NMR ofProteins and Nucleic Acids, John Wiley, New York, N.Y. (1986). NMRtechniques further include, but are not limited to, (i)Reduced-dimensionality (RD) NMR (Szyperski et al., Proc. Natl. Acad.Sci. U.S.A., 99:8009-8014 (2002)); (ii) G-matrix FT (GFT) projection NMR(Atreya et al., J. Am. Chem. Soc., 127:4554-4555 (2005); Eletsky et al.,J. Am. Chem. Soc., 127:14578-14579 (2005); Yang et al., J. Am. Chem.Soc., 127:9085-9099 (2005); Szyperski et al., Magn. Reson. Chem.,44:51-60 (2006); Atreya et al., J. Am. Chem. Soc., 129:680-692 (2007);Kupce et al., J. Am. Chem. Soc., 126:6429-40 (2004); Hiller et al.,Proc. Natl. Acad. Sci. U.S.A., 102:10876-10881 (2005); and Eghbalnia etal., J. Am. Chem. Soc., 127: 12528-12536 (2005)); and (iii) CovarianceNMR spectroscopy (Bruschweiler, J. Chem. Phys., 121:409-414 (2004);Zhang et al., J. Am. Chem. Soc., 126:13180-13181 (2004); and Chen etal., J. Am. Chem. Soc., 128:15564-15565 (2006)). These publications areincorporated by reference herein in their entireties.

Thus, as used herein, the term nuclear magnetic resonance (NMR)encompasses a variety of methods including but not limited to,one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR), correlationspectroscopy NMR (COSY-NMR), total correlated spectroscopy NMR(TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR),heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclearoverhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhausereffect spectroscopy (NOESY-NMR), transverse relaxation optimizedspectroscopy (TROSY-NMR) and combinations thereof. For more descriptionon TROSY-NMR see Pervushin, et al., “Attenuated T₂ relaxation by mutualcancellation of dipole-dipole coupling and chemical shift anisotropyindicates an avenue to NMR structures of very large biologicalmacromolecules in solution” PNAS 94:12366-71 (1997), which isincorporated by reference herein in its entirety.

As used herein, the term “chemical shift,” in nuclear magnetic resonance(NMR) spectroscopy, refers to the resonant frequency of a nucleusrelative to a standard or baseline. Some atomic nuclei possess amagnetic moment (nuclear spin), which gives rise to different energylevels and resonance frequencies in a magnetic field. The electrondistribution of the same type of nucleus (e.g. ¹H, ¹³C, ¹⁵N) usuallyvaries according to the local geometry and with it the local magneticfield at each nucleus. This is reflected in the spin energy levels (andresonance frequencies). The variation of nuclear magnetic resonancefrequencies of the same kind of nucleus, due to variations in theelectron distribution, is called the chemical shift. The size of thechemical shift is typically given with respect to a reference frequencyor reference sample usually a molecule with a barely distorted electrondistribution. Typically, a ¹H-¹⁵N HSQC spectrum is used to obtainchemical shift values. However, as provided in the methods herein, anyNMR analysis method can be used.

As used herein, the term “chemical shift of an amino acid” includes thechemical shift of an element within the amino acid, e.g., H, C or N. Asused herein, the term “element” refers to an atom distinguished by itsatomic number, which is the number of protons in its nucleus. Exemplaryelements include, but are not limited to, H (hydrogen), N (nitrogen) andC (carbon).

Exemplary chemical shift values for certain amino acids in the SENP1polypeptide are shown in Table 3 and exemplary chemical shift values forcertain amino acids in the SENP1 polypeptide when bound to SUMO areshown in Table 4. The sample conditions that correlate to the chemicalshifts listed in Table 3 are 20 mM sodium phosphate, at pH 6.8 at 25° C.The sample conditions that correlate to the chemical shifts listed inTable 4 are 20 mM sodium phosphate and containing 150 mM NaCl, at pH 7,at 35° C. The values of the chemical shifts listed in Table 3 and Table4 may vary by as much as 1 ppm for 41, and as much as 5 ppm for ¹⁵N and¹³C due to differences in experimental conditions such as sample pH,temperature, addition of other components (e.g., salt), or amino acidsubstitutions in SENP1 and/or SUMO that may affect the function of SENP1and/or SUMO. Thus, the chemical shifts listed in Tables 3 and 4 may varyfrom 1 ppm for ¹H and from 5 ppm for ¹⁵N and ¹³C.

Thus, the peaks or chemical shifts in Tables 3 and 4 can be used bythose of skill in the art to determine whether a test compound bindsSENP1 by correlating experimental peaks or chemical shifts to thoseprovided in Tables 3 and 4. For example, the peaks or chemical shiftsobtained by NMR in the presence of a test compound can be compared tothe corresponding peaks or chemical shifts in Tables 3 or 4 to determinewhether the test compound binds SENP1. Thus, the chemical shift for anamino acid of SENP1 in Table 3 or 4 can be compared to the correspondingchemical shift obtained for the same amino acid in SENP1 in the presenceof a test compound. When performing such comparisons, one of skill inthe art will account for variances known to affect chemical shift valuesdue to changes in experimental conditions, e.g., pH, temperature,addition of other components (e.g., salt), or amino acid substitutions.In some embodiments, detection of a change of greater than 5 ppm in thechemical shift for ¹⁵N or ¹³C of an amino acid of SENP1 or greater than1 ppm in the chemical shift for ¹H of an amino acid of SENP1 indicatesnon-correlation of peaks. Optionally, the change is as compared to thecorresponding chemical shift value for ¹⁵N, ¹³C, or ¹H of an amino acidof SENP1 in Table 3 or Table 4.

As used herein, the binding of a compound to SENP1 may be selective. Theterms “selectively binds,” “selectively binding,” or “specificallybinding” refers to the compound binding SENP1 to the partial or completeexclusion of other agents or compounds. By binding is meant a detectablebinding, for example, binding above the background of the assay method.Optionally, detectable binding is evidenced by comparing baseline toexperimental values, e.g., by comparing baseline NMR data (e.g.,chemical shift values or digital resolution spectra) to experimental NMRdata (e.g., chemical shift values or digital resolution spectra). Thus,binding can be determined by detecting changes or perturbations in anNMR measurement or spectrum for one sample, e.g., a control sample,compared to another or second sample, e.g., a sample containing a testcompound. Detectable changes or perturbations in NMR signals includechanges in location (chemical shift). General NMR techniques forproteins, including multidimensional NMR experiments and determinationof protein-ligand interactions can be found in David G. Reid (ed.),Protein NMR Techniques, Humana Press, Totowa N.J. (1997). By way ofexample, detection of a perturbation or change includes detection of adifference in the chemical shift of SENP1 or SENP1-SUMO complex in thepresence of a compound as compared to the chemical shift in the absenceof the compound. The perturbation or change (whether increased ordecreased) can include significant differences in an NMR measurement orspectrum (e.g., chemical shift) and can be greater than the experimentalerror or greater than the error bar range. For example, a change of atleast about 1.1 times of the digital resolution of a spectrum orchemical shift for one or more amino acid residues of SENP1 in thepresence of a compound can indicate the compound binds SENP1. Thus, achange of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 10, 20 times or more of the digital resolution of an NMRmeasurement or spectrum, e.g., chemical shift, observed in the presenceof a compound as compared to a control can indicate the compound bindsSENP1.

The terms greater, higher, increases, elevates, or elevation refer toincreases above a control. The terms low, lower, reduces, or reductionrefer to any decrease below control levels. For example, control levelsare levels prior to, or in the absence of, addition of a compound.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Acontrol can also represent an average value gathered from a number oftests or results. One of skill in the art will recognize that controlscan be designed for assessment of any number of parameters. For example,a control can be devised to compare therapeutic benefit based onpharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of side effects). One of skill in the art will understandwhich controls are valuable in a given situation and be able to analyzedata based on comparisons to control values. Controls are also valuablefor determining the significance of data. For example, if values for agiven parameter are widely variant in controls, variation in testsamples will not be considered as significant.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more identity over a specified region, e.g.,of the entire polypeptide sequences of the invention or individualdomains of the polypeptides of the invention), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Such sequencesare then said to be “substantially identical.” This definition alsorefers to the complement of a test sequence. Optionally, the identityexists over a region that is at least about 10 to about 100, about 20 toabout 75, about 30 to about 50 amino acids or nucleotides in length.Optionally, the identity exists over a region that is at least about 50amino acids in length, or more preferably over a region that is 100 to500 or 1000 or more amino acids in length. The present inventionincludes polypeptides that are substantially identical to any of SEQ IDNOs:1, 2, 3, 4, 5, 6, or 7.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information, asknown in the art. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

An amino acid residue in a polypeptide “corresponds” to or “iscorresponding to” a given residue when it occupies the same essentialstructural position within the polypeptide as the given residue. Forexample, a selected residue in a comparison polypeptide corresponds toposition 603 in a polypeptide provided herein (e.g., a SENP1polypeptide), when the selected residue occupies the same essentialspatial or structural relationship to position 603 as assessed usingapplicable methods in the art. For example, a comparison polypeptide maybe aligned for maximum sequence homology with the polypeptide providedherein and the position in the aligned comparison polypeptide thataligns with position 603 may be determined to correspond to it.Alternatively, instead of (or in addition to) a primary sequencealignment as described above, a three dimensional structural alignmentcan also be used, e.g., where the structure of the comparisonpolypeptide is aligned for maximum correspondence with a polypeptideprovided herein and the overall structures compared. In this case, anamino acid that occupies the same essential position as position 603 inthe structural model may be said to correspond.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The “active-site” of a protein or polypeptide refers to a protein domainthat is structurally, functionally, or both structurally andfunctionally, active. For example, the active-site of a protein can be asite that catalyzes an enzymatic reaction, i.e., a catalytically activesite. An active site refers to a domain that includes amino acidresidues involved in binding of a substrate for the purpose offacilitating the enzymatic reaction. Optionally, the term active siterefers to a protein domain that binds to another agent, molecule orpolypeptide. For example, the active sites of SENP1 include sites onSENP1 that bind to or interact with SUMO. A protein may have one or moreactive-sites.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al., John Wiley& Sons.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

The term “pharmaceutically acceptable salts” or “pharmaceuticallyacceptable carrier” is meant to include salts of the active compoundswhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the compounds described herein.When compounds of the present application contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable base addition salts include sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present application containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts includethose derived from inorganic acids like hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19(1977)). Other pharmaceutically acceptable carriers known to those ofskill in the art are suitable for compositions of the presentapplication.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating afluorescent label into a peptide specifically reactive with a targetpeptide (e.g., SENP1 polypeptide, SUMO protein or test compound). Inembodiments, the label is a fluorescent label. Any method known in theart for conjugating a polypeptide to the label may be employed, e.g.,using methods described in Hermanson, Bioconjugate Techniques 1996,Academic Press, Inc., San Diego.

A “labeled protein or polypeptide” is one that is bound, eithercovalently, through a linker or a chemical bond, or noncovalently,through ionic, van der Waals, electrostatic, or hydrogen bonds to alabel such that the presence of the labeled protein or polypeptide maybe detected by detecting the presence of the label bound to the labeledprotein or polypeptide.

Methods

Provided herein are methods of detecting binding of an SENP1 polypeptideto a compound. The method includes the steps of contacting an SENP1polypeptide with a compound, allowing the compound to bind to the SENP1polypeptide, thereby forming a SENP1-compound complex, and detecting theSENP1-compound complex using nuclear magnetic resonance, therebydetecting binding of the SENP1 polypeptide to the compound.

A “compound” as provided herein refers to a polypeptide, protein, aminoacid, small molecule or chemical compound that is capable of binding aSENP1 polypeptide or fragment thereof. In embodiments, the compoundbinds a SENP1 protein of SEQ ID NO:1, 2, 3, 4, 5, 6, or 7. Inembodiments, the compound is a modulator of SENP1 activity. Inembodiments, the compound is an inhibitor of SENP1 activity. Inembodiments, the compound is an activator of SENP1 activity. Inembodiments, the compound is a small molecule. A small molecule asprovided herein include, but are not limited to the compounds in Tables1 and 2 and those described in WO 2012/064887, which is incorporated byreference herein in its entirety. As used herein, the term “smallmolecule” refers to an organic compound containing carbon. A smallmolecule is generally, but not necessarily, of low molecular weight,e.g., less than 1000 Daltons.

A “test compound” as provided herein refers to a compound useful for thescreening methods provided herein. A test compound may be capable ofbinding a SENP1 polypeptide or fragment thereof as provided herein. Inembodiments, the test compound binds a SENP1 polypeptide or fragmentthereof. In embodiments, the binding of the test compound to the SENP1polypeptide or fragment thereof is detected by nuclear magneticresonance. In embodiments, the test compound does not bind a SENP1polypeptide or fragment thereof.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a compound or protein-inhibitor interactionmeans negatively affecting (e.g., decreasing) the activity or functionof the protein (e.g. decreasing gene transcription or translation)relative to the activity or function of the protein in the absence ofthe inhibitor. In embodiments, inhibition refers to reduction of adisease or symptoms of disease (e.g., cancer). In embodiments,inhibition refers to a reduction in the activity of an enzymaticactivity (e.g., SENP activity). In embodiments, inhibition refers to areduction in the activity of a signal transduction pathway or signalingpathway (e.g. cell cycle). Thus, inhibition includes, at least in part,partially or totally blocking stimulation, decreasing, preventing, ordelaying activation, or inactivating, desensitizing, or down-regulatingtranscription, translation, signal transduction or enzymatic activity orthe amount of a protein (e.g. a cellular protein or a viral protein). Inembodiments, inhibition refers to inhibition of SENP1.

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator”interchangeably refer to a substance that results in a detectably lowerexpression or activity level as compared to a control. The inhibitedexpression or activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or less than that in a control. In certain instances, the inhibitionis 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more incomparison to a control. An “inhibitor” is a siRNA, (e.g., shRNA, miRNA,snoRNA), compound or small molecule that inhibits cellular function(e.g., replication) e.g., by binding, partially or totally blockingstimulation, decrease, prevent, or delay activation, or inactivate,desensitize, or down-regulate signal transduction, gene expression orenzymatic activity necessary for protein activity. Inhibition asprovided herein may also include decreasing or blocking a proteinactivity (e.g., activity of SENP1).

The terms “agonist,” “activator,” “upregulator,” etc. refer to asubstance capable of detectably increasing the expression or activity ofa given gene or protein. The agonist can increase expression or activity10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to acontrol in the absence of the agonist. In certain instances, expressionor activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold orhigher than the expression or activity in the absence of the agonist.

Optionally, the compound is a small molecule. Optionally, the step ofdetecting includes detecting a perturbation in the presence of thecompound relative to the absence of the compound. For example, bindingof a compound to SENP1 is detected if a perturbation is detected in anNMR measurement or spectrum in the presence of the compound as comparedto or relative to the absence of the compound. Optionally, the step ofdetecting includes determining a chemical shift for an amino acid in anactive site of the SENP1 polypeptide. Binding is detected by a change inthe chemical shift in the presence of the compound relative to thecorresponding chemical shift in the absence of the compound. Optionally,the active site is a catalytically active site. Optionally, the activesite is a site involved in SUMO binding, e.g., the active site is a siteon SENP1 that binds to the SUMO protein. Thus, the step of detectingincludes determining a chemical shift for an amino acid involved inbinding of SENP1 polypeptide to SUMO. Optionally, the chemical shift isdetermined for one or more amino acids of SEQ ID NOs:3, 4, 5, 6 or 7.

Optionally, the chemical shift is determined for one or more amino acidresidues selected from the group consisting of D550, H533, C603, W465,W534, L466, G531, C535, M552, G554, E469 and Q596 of SEQ ID NO:1.

In embodiments, the change is a change in the chemical shift of aminoacid residue D550, H533, C603, W465, W534, L466, G531, C535, M552, G554,E469 or Q596 of SEQ ID NO:1. In embodiments, the change is a change inthe chemical shift of amino acid residue D550, H533, C603, W465, W534,L466, G531, C535, M552, G554, E469 and Q596 of SEQ ID NO:1. Inembodiments, the change is a change in the chemical shift of amino acidresidue D550 of SEQ ID NO:1. In embodiments, the change is a change inthe chemical shift of amino acid residue H533 of SEQ ID NO:1. Inembodiments, the change is a change in the chemical shift of amino acidresidue C603 of SEQ ID NO:1. In embodiments, the change is a change inthe chemical shift of amino acid residue W465 of SEQ ID NO:1. Inembodiments, the change is a change in the chemical shift of amino acidresidue W534 of SEQ ID NO:1. In embodiments, the change is a change inthe chemical shift of amino acid residue L466 of SEQ ID NO:1. Inembodiments, the change is a change in the chemical shift of amino acidresidue G531 of SEQ ID NO:1. In embodiments, the change is a change inthe chemical shift of amino acid residue C535 of SEQ ID NO:1. Inembodiments, the change is a change in the chemical shift of amino acidresidue M552 of SEQ ID NO:1. In embodiments, the change is a change inthe chemical shift of amino acid residue G554 of SEQ ID NO:1. Inembodiments, the change is a change in the chemical shift of amino acidresidue E469 of SEQ ID NO:1. In embodiments, the change is a change inthe chemical shift of amino acid residue Q596 of SEQ ID NO:1.

In embodiments, the change is a change in the chemical shift of an aminoacid residue corresponding to D550, H533, C603, W465, W534, L466, G531,C535, M552, G554, E469 or Q596 of SEQ ID NO:1. In embodiments, thechange is a change in the chemical shift of an amino acid residuecorresponding to D550, H533, C603, W465, W534, L466, G531, C535, M552,G554, E469 and Q596 of SEQ ID NO:1. In embodiments, the change is achange in the chemical shift of an amino acid residue corresponding toD550 of SEQ ID NO:1. In embodiments, the change is a change in thechemical shift of an amino acid residue corresponding to H533 of SEQ IDNO:1. In embodiments, the change is a change in the chemical shift of anamino acid residue corresponding to C603 of SEQ ID NO:1. In embodiments,the change is a change in the chemical shift of an amino acid residuecorresponding to W465 of SEQ ID NO:1. In embodiments, the change is achange in the chemical shift of an amino acid residue corresponding toW534 of SEQ ID NO:1. In embodiments, the change is a change in thechemical shift of an amino acid residue corresponding to L466 of SEQ IDNO:1. In embodiments, the change is a change in the chemical shift of anamino acid residue corresponding to G531 of SEQ ID NO:1. In embodiments,the change is a change in the chemical shift of an amino acid residuecorresponding to C535 of SEQ ID NO:1. In embodiments, the change is achange in the chemical shift of an amino acid residue corresponding toM552 of SEQ ID NO:1. In embodiments, the change is a change in thechemical shift of an amino acid residue corresponding to G554 of SEQ IDNO:1. In embodiments, the change is a change in the chemical shift of anamino acid residue corresponding to E469 of SEQ ID NO:1. In embodiments,the change is a change in the chemical shift of an amino acid residuecorresponding to Q596 of SEQ ID NO:1.

In embodiments, the SENP1 polypeptide includes amino acid residue 603 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 603 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 550 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 550 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 533 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 533 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 465 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 465 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 534 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 534 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 466 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 466 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 531 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 531 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 535 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 535 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 552 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 552 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 554 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 554 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 469 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 469 of SEQ ID NO:1. Inembodiments, the SENP1 polypeptide includes amino acid residue 596 ofSEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an aminoacid residue corresponding to amino acid residue 596 of SEQ ID NO:1.

Optionally, the chemical shift is determined for a mutation at aminoacid residue 603 of SEQ ID NO:1. Optionally, the mutation is C603S.Optionally, the chemical shift is determined for one or more amino acidresidues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQID NO:1. Optionally, the SENP1 polypeptide or SENP1-compound complex isbound to a SUMO protein thereby forming a SENP1-SUMO complex orSENP1-SUMO-compound complex. Optionally, the SUMO protein is a truncatedSUMO protein. Optionally, the compound does not interact with C603 ofSEQ ID NO:1 of SENP1, e.g., the compound does not covalently modify C603of SENP1. Thus, the provided methods optionally include detectingbinding by producing an NMR spectra of the SENP-1 compound complex andidentifying a change in the NMR spectra relative to the absence of thecompound. Optionally, the change is a change in the chemical shift of anamino acid of SEQ ID NOs:3, 4, 5, 6 or 7. Optionally, the change is achange in the chemical shift of an amino acid selected from the groupconsisting of D550, H533, C603, W465, W534, L466, G531, C535, M552,G554, E469 and Q596. Optionally, the change is a change in the chemicalshift of the amino acid S603. Optionally, the change is a change in thechemical shift of an amino acid residue 440-455, 463-473, 493-515,529-535, 550-554, or 596-603 of SEQ ID NO:1.

Also provided is a method of screening for compounds that bind SENP1including the steps of providing a first sample comprising SENP1 or anSENP1-SUMO complex, determining an NMR spectra of the first sample,providing a second sample comprising an SENP1-compound complex or anSENP1-SUMO-compound complex, and determining an NMR spectra of thesecond sample. Detection of a change in the NMR spectra in the secondsample as compared to the first sample indicates the compound bindsSENP1.

Provided are methods of screening for an inhibitor of SENP1. The methodsinclude contacting a composition comprising an SENP1 polypeptide with atest compound and detecting whether the test compound binds the SENP1polypeptide or fragment thereof by nuclear magnetic resonance.

Optionally, the step of detecting includes detecting a perturbation inthe presence of the compound relative to the absence of the compound.For example, the test compound binds or inhibits SENP1 if a perturbationis detected in an NMR measurement or spectrum in the presence of thecompound as compared to or relative to the absence of the compound.Optionally, the step of detecting comprises determining a chemical shiftfor one or more amino acids in the active site of the SENP1 polypeptide.The chemical shift in the presence of the compound will be changedrelative to the corresponding chemical shift in the absence of the testcompound if the test compound binds to SENP1. Optionally, the activesite is a catalytically active site. Optionally, the active site is asite involved in SUMO binding, e.g., the active site is a site on SENP1that binds to the SUMO protein. Thus, the step of detecting includesdetermining a chemical shift for an amino acid involved in binding ofSENP1 polypeptide to SUMO. Optionally, the chemical shift is determinedfor one or more amino acids of SEQ ID NOs:3, 4, 5, 6 OR 7. Optionally,the chemical shift is determined for one or more amino acid residuesselected from the group consisting of D550, H533, C603, W465, W534,L466, G531, C535, M552, G554, E469 and Q596 of SEQ ID NO:1. Optionally,the chemical shift is determined for a mutation at amino acid residue603 of SEQ ID NO:1. Optionally, the mutation is C603S. Optionally, thechemical shift is determined for one or more amino acid residues440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.Optionally, the SENP1 polypeptide is bound to a SUMO protein therebyforming a SENP1-SUMO complex. Optionally, the SUMO protein is atruncated SUMO protein. Optionally, the composition comprising the SENP1polypeptide or SENP1-SUMO complex is an aqueous solution. Optionally,the composition is at a pH from about 6.0 to about 7.5. Optionally, thepH is about 6.8. Optionally, the composition comprises a bufferingagent, a reducing agent, a base or combinations thereof. Optionally, thecomposition comprises sodium phosphate, D₂O, sodium azide,dithiothreitol or combinations thereof. The sodium phosphate can bepresent at about 20 mM. Optionally, the compound to be tested is a smallmolecule. Optionally, the compound does not interact with C603 numberedrelative to SEQ ID NO:1 of SENP1, e.g., the compound does not covalentlymodify C603 of SENP1. Optionally, in the provided methods, the SENP1binds the compound forming an SENP1-compound complex and the detectingcomprises producing an NMR spectra of the SENP1-compound complex andidentifying a change in the NMR spectra relative to the absence of thecompound. Optionally, the change is a change in the chemical shift of anamino acid of SEQ ID NOs:3, 4, 5, 6 or 7. Optionally, the change is achange in the chemical shift of an amino acid selected from the groupconsisting of D550, H533, C603, W465, W534, L466, G531, C535, M552,G554, E469 and Q596. Optionally, the change is a change in the chemicalshift of the amino acid S603. Optionally, the change is a change in thechemical shift of an amino acid residue 440-455, 463-473, 493-515,529-535, 550-554, or 596-603 of SEQ ID NO:1. Optionally, the change is achange in the chemical shift of an amino acid in the active site ofSENP1. Optionally, the active site is a catalytically active site or asite that binds to the SUMO protein.

Also provided are methods of identifying an SENP1 inhibitor that includecombining an SENP1 polypeptide, a SUMO protein, and a test compound in areaction vessel, allowing the SENP1 polypeptide, SUMO protein and testcompound to form a SENP1-SUMO-compound complex, and detecting theSENP1-SUMO-compound complex thereby identifying the compound as a SENP1inhibitor. A “reaction vessel” as provided herein refers to a vial,tube, flask, bottle, syringe or other container means, into which theSENP1 polypeptide, SUMO protein and test compound are combined to allowthe formation of a SENP1-SUMO-compound complex.

Optionally, one or more of the SENP1 polypeptide, SUMO protein or testcompound is labeled. Optionally, the label is a fluorescent label.Optionally, the test compound comprises a fluorescent label. Optionally,the SUMO is a truncated SUMO protein. Optionally, the SUMO comprisesamino acid residues 1-92 of the SUMO protein. Optionally, the SUMOprotein comprises SEQ ID NO:8 or SEQ ID NO:9. Optionally, the SENP1polypeptide comprises SEQ ID NOs:1, 2, 3, 4, 5, 6, or 7. Optionally, theSENP1 polypeptide comprises amino acid residue 603 of SEQ ID NO:1.Optionally, the SENP1 polypeptide comprises a mutation at amino acidresidue 603 of SEQ ID NO:1. Optionally, the mutation is C603S.Optionally, the SENP1 polypeptide comprises amino acid residues 440-455,463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.Optionally, the test compound is a small molecule. In the providedmethods, the detecting can be performed by a variety of methods known tothose skilled in the art and described in the example below. See, e.g.,Protein-Ligand Interactions, Vol. 1008, Methods in Molecular Biology,Humana Press, Inc., Clifton, N.J., Williams and Daviter, Eds. (2013).For example, a wide variety of assays for detecting binding can be usedincluding labeled in vitro protein-ligand binding assays, cell basedassays, immunoassays, and the like. Optionally, detecting can beperformed using solution-phase binding assays, e.g., fluorescentpolarization. Thus, binding can be detected by fluorescent polarization(Rossi et al., Nat. Protoc. 6(3):365-87 (2011)). Optionally, binding isdetected by detecting a change in the thermal properties of SENP1, e.g.,the thermal property can be the melting temperature of SENP1. In someembodiments, the detecting is performed using nuclear magneticresonance. Optionally, the detecting comprises producing an NMR spectraof the SENP1-SUMO-compound complex and identifying a change in the NMRspectra relative to the absence of the test compound. Optionally, thechange is a change in the chemical shift of an amino acid in an activesite of the SENP1 polypeptide. The active site can be, for example, acatalytically active site or a site that binds to the SUMO protein.Optionally, the amino acid is an amino acid of SEQ ID NOs:3, 4, 5, 6 OR7. Optionally, the amino acid is selected from the group consisting ofD550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 andQ596. Optionally, the amino acid is S603. Optionally, the amino acid isamino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or596-603 of SEQ ID NO:1.

As used throughout, the term “SENP1 polypeptide” refers to full lengthSENP1 and fragments thereof. The sequence and structure of the SENP1polypeptide is known. (See above and Protein Data Bank (PDB) accessioncodes 2IYC and 2IY1; Shen et al., Nat. Struct. Mol. Biol.13(12):1069-1077 (2006); and Xu et al., Biochem. J. 398(3):345-52(2006)). Optionally, the SENP1 polypeptide comprises SEQ ID NOs:1, 2, 3,4, 5, 6, or 7. Optionally, the SENP1 polypeptide comprises amino acidresidue 603 of SEQ ID NO:1. Optionally, the SENP1 polypeptide comprisesa mutation at amino acid residue 603 of SEQ ID NO:1. Optionally, themutation is C603S. Optionally, the SENP1 polypeptide comprises aminoacid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 ofSEQ ID NO:1.

Optionally, in the provided methods, SENP1 is bound to SUMO or afragment thereof, e.g., a truncated SUMO protein. Thus, optionally, theSENP1 is bound to a SUMO protein thereby forming a SENP1-SUMO complex.Optionally, the SUMO protein is a truncated SUMO protein. Optionally,the SUMO protein is SEQ ID NO:8 or 9. As used herein, the term“truncated SUMO protein” refers to a SUMO protein or polypeptide thathas been manipulated to remove at least one amino acid residue relativeto wild-type SUMO, e.g., a SUMO protein or polypeptide that occurs innature. Exemplary wild-type SUMO proteins include, but are not limitedto, SEQ ID NO:9 and those found at GenBank Accession Nos. AAC50996.1,NP_008868.3, NP_001005849.1, P55854.2, and NP_008867.2. Truncated SUMOproteins include, but are not limited to, SEQ ID NO:8. As used herein,the term “SUMO” refers to SUMO1, SUMO2, or SUMO3 or fragments thereof orcomplexes thereof, e.g., SUMO2/3. The nucleic acid and amino acidsequences for SUMO are known. See, for example, Hay, Mol. Cell18(1):1-12 (2005); and Yeh, et al., J. Biol. Chem., 284(13):8223-7(2009). For example, nucleic acid and amino acid sequences for SUMO-1can be found at GenBank Accession Nos. U67122.1 and AAC50996.1. Nucleicacid and amino acid sequences for SUMO-2 can be found at GenBankAccession Nos. NM_006937.3, NM_001005849.1, NP_008868.3 andNP_001005849.1. Nucleic acid and amino acid sequences for SUMO-3 can befound at GenBank Accession Nos. NM_006936.2, P55854.2, and NP_008867.2.Optionally, the SENP1 is bound to SUMO1 to form an SENP1-SUMO1 complex.

The provided SENP1 polypeptides and/or SUMO polypeptides and fragmentsthereof may contain one or more modifications, e.g., a conservativemodification. As used herein, the term “modification” refers to amodification in a nucleic acid sequence of a gene or an amino acidsequence. Modifications include, but are not limited to, insertions,substitutions and deletions. Amino acid sequence modifications typicallyfall into one or more of three classes: substitutional, insertional, ordeletional modifications. Insertions include amino and/or terminalfusions as well as intrasequence insertions of single or multiple aminoacid residues. Insertions ordinarily will be smaller insertions thanthose of amino or carboxyl terminal fusions, for example, on the orderof one to four residues. Deletions are characterized by the removal ofone or more amino acid residues from the protein sequence. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once. Substitutions, deletions,insertions or any combination thereof may be combined to arrive at afinal construct. Substitutional modifications are those in which atleast one residue has been removed and a different residue inserted inits place.

Modifications are generated using any number of methods known in theart. For example, site directed mutagenesis can be used to modify anucleic acid sequence. One of the most common methods of site-directedmutagenesis is oligonucleotide-directed mutagenesis. Inoligonucleotide-directed mutagenesis, an oligonucleotide encoding thedesired change(s) in sequence is annealed to one strand of the DNA ofinterest and serves as a primer for initiation of DNA synthesis. In thismanner, the oligonucleotide containing the sequence change isincorporated into the newly synthesized strand. See, for example,Kunkel, 1985, Proc. Natl. Acad. Sci. USA, 82:488; Kunkel et al., 1987,Meth. Enzymol., 154:367; Lewis & Thompson, 1990, Nucl. Acids Res.,18:3439; Bohnsack, 1996, Meth. Mol. Biol., 57:1; Deng & Nickoloff, 1992,Anal. Biochem., 200:81; and Shimada, 1996, Meth. Mol. Biol., 57:157.Other methods are routinely used in the art to introduce a modificationinto a sequence. For example, modified nucleic acids are generated usingPCR or chemical synthesis, or polypeptides having the desired change inamino acid sequence can be chemically synthesized. See, for example,Bang & Kent, 2005, Proc. Natl. Acad. Sci. USA, 102:5014-9 and referencestherein.

Also provided herein are nucleic acids encoding the polypeptidesdescribed throughout. It is understood that the nucleic acids that canencode those peptide, polypeptide, or protein sequences, variants andfragments thereof are also disclosed. This would include all degeneratesequences related to a specific polypeptide sequence, i.e. all nucleicacids having a sequence that encodes one particular polypeptide sequenceas well as all nucleic acids, including degenerate nucleic acids,encoding the disclosed variants and derivatives of the polypeptidesequences. Thus, while each particular nucleic acid sequence may not bewritten out herein, it is understood that each and every sequence is infact disclosed and described herein through the disclosed polypeptidesequence.

Provided herein are compounds to be tested for their ability to bindand/or inhibit SENP1. As used herein, an inhibitor refers to an agent orcompound that inhibits SENP1 directly or indirectly. For example, aninhibitor of SENP1 can inhibit the expression or activity of SENP1.Compounds to be tested in the provided methods include, but are notlimited to, small molecules, peptides, nucleic acids and antibodies.Optionally, the compound to be tested is a small molecule. Optionally,the small molecule is an inhibitor of SENP1. Small molecule inhibitorsof SENP1 include, but are not limited to the compounds in Tables 1 and 2and those described in WO 2012/064887, which is incorporated byreference herein in its entirety. As used herein, the term “smallmolecule” refers to an organic compound containing carbon. A smallmolecule is generally, but not necessarily, of low molecular weight,e.g., less than 1000 Daltons.

Once a compound has been identified as binding to SENP1 and/orinhibiting SENP1, the compound can be further tested for its bindingand/or inhibitory abilities using a variety of known methods includingthe methods described in the example below. Various assays fordetermining levels and activities of protein are available, such asamplification/expression methods, immunohistochemistry methods, FISH andshed antigen assays, southern blotting, or PCR techniques. Moreover, theprotein expression or amplification may be evaluated using in vivodiagnostic assays.

Compositions and Apparatuses for NMR Analysis

Provided herein are compositions comprising a SENP1 polypeptide and NMRapparatuses comprising the compositions for NMR analysis. Optionally,the composition is an aqueous solution. Optionally, the aqueous solutioncomprises an SENP1 polypeptide at a pH from about 6.0 to about 7.5. Forexample, the pH can be about 6.8. The provided compositions or aqueoussolutions can further include, for example, buffering agents, reducingagents, solvents, bases and combinations thereof. Buffering agentsinclude, but are not limited to, phosphate or citrate buffers. Reducingagents include but are not limited to, dithiothreitol, and sodiumborohydride. Bases include, but are not limited to, metal oxides andsalts of carbanions, amides and hydrides. Solvents include, but are notlimited to, dimethyl sulfoxide (DMSO) Optionally, the compositions caninclude sodium phosphate, DMSO, D₂O, sodium azide, dithiothreitol orcombinations thereof. By way of example, the sodium phosphate can bepresent at about 20 mM. Optionally, the SENP1 polypeptide is bound to aSUMO protein thereby forming a SENP1-SUMO complex. Optionally, the SENP1polypeptide is bound to a compound thereby forming a SENP1-compoundcomplex. Optionally, the SENP1 polypeptide is bound to a SUMO proteinthereby forming a SENP1-SUMO-compound complex. Optionally, the SUMOprotein is a truncated SUMO protein. Optionally, the SENP1 polypeptidecomprises SEQ ID NO:1, 2, 3, 4, 5, 6, or 7. Optionally, the SENP1polypeptide comprises amino acid residues 440-455, 463-473, 493-515,529-535, 550-554, or 596-603 numbered relative to SEQ ID NO:1.

An NMR apparatus comprising an NMR sample container for NMR analysis,said NMR sample container comprising the aqueous composition or solutionis also provided. NMR apparatuses are known and can be obtained fromcommercially available sources. Makers of NMR equipment include, but arenot limited to, Bruker (Germany), Oxford Instruments (United Kingdom),General Electric (Fairfield, Conn.), Philips (Amsterdam, Netherlands),Siemens AG (Munich, Germany) and Agilent Technologies, Inc. (SantaClara, Calif.).

Compositions

Provided herein are compositions including the inhibitors identified bythe screening and binding methods provided herein. The compositions are,optionally, suitable for formulation and administration in vitro or invivo. Optionally, the compositions comprise one or more of the providedagents and a pharmaceutically acceptable carrier. Suitable carriers andtheir formulations are described in Remington: The Science and Practiceof Pharmacy, 21^(st) Edition, David B. Troy, ed., Lippicott Williams &Wilkins (2005). By pharmaceutically acceptable carrier is meant amaterial that is not biologically or otherwise undesirable, i.e., thematerial is administered to a subject without causing undesirablebiological effects or interacting in a deleterious manner with the othercomponents of the pharmaceutical composition in which it is contained.If administered to a subject, the carrier is optionally selected tominimize degradation of the active ingredient and to minimize adverseside effects in the subject.

The inhibitors are administered in accord with known methods, such asintravenous administration, e.g., as a bolus or by continuous infusionover a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, intratumoral or inhalation routes. Theadministration may be local or systemic. The compositions can beadministered via any of several routes of administration, includingtopically, orally, parenterally, intravenously, intra-articularly,intraperitoneally, intramuscularly, subcutaneously, intracavity,transdermally, intrahepatically, intracranially,nebulization/inhalation, or by installation via bronchoscopy. Thus, thecompositions are administered in a number of ways depending on whetherlocal or systemic treatment is desired, and on the area to be treated.

The compositions for administration will commonly comprise an agent asdescribed herein (e.g. inhibitor of SENP1) dissolved in apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., buffered saline and thelike. These solutions are sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like. Theconcentration of active agent in these formulations can vary widely, andwill be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the subject's needs.

Pharmaceutical formulations, particularly, of the modified viruses canbe prepared by mixing the modified adenovirus (or one or more nucleicacids encoding the modified adenovirus) having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients orstabilizers. Such formulations can be lyophilized formulations oraqueous solutions.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations used. Acceptable carriers,excipients or stabilizers can be acetate, phosphate, citrate, and otherorganic acids; antioxidants (e.g., ascorbic acid) preservatives lowmolecular weight polypeptides; proteins, such as serum albumin orgelatin, or hydrophilic polymers such as polyvinylpyllolidone; and aminoacids, monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents; and ionic and non-ionicsurfactants (e.g., polysorbate); salt-forming counter-ions such assodium; metal complexes (e. g. Zn-protein complexes); and/or non-ionicsurfactants. The modified adenovirus (or one or more nucleic acidsencoding the modified adenovirus) can be formulated at any appropriateconcentration of infectious units.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the modified adenovirussuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The inhibitors of SENP1 can be made into aerosol formulations (i.e.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the provided methods, compositions can beadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically intratumorally, or intrathecally.Parenteral administration, intratumoral administration, and intravenousadministration are the preferred methods of administration. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced or infected by adenovirus or transfected with nucleic acidsfor ex vivo therapy can also be administered intravenously orparenterally as described above.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. Thus, the pharmaceuticalcompositions can be administered in a variety of unit dosage formsdepending upon the method of administration. For example, unit dosageforms suitable for oral administration include, but are not limited to,powder, tablets, pills, capsules and lozenges.

Methods of Treatment

The provided inhibitors of SENP1 can be administered for therapeutic orprophylactic treatments or used in the laboratory. Thus, provided is amethod of treating a proliferative disorder in a subject. The methodincludes administering the provided inhibitors of SENP1 or compositionsto the subject. As described throughout, the pharmaceutical compositionis administered in any number of ways including, but not limited to,intravenously, intravascularly, intrathecally, intramuscularly,subcutaneously, intraperitoneally, or orally. Optionally, the methodfurther comprising administering to the subject one or more additionaltherapeutic agents. Optionally, the therapeutic agent is achemotherapeutic agent.

As described throughout, the proliferative disorder can be cancer.Optionally, the proliferative disorder is selected from the groupconsisting of lung cancer, prostate cancer, colorectal cancer, breastcancer, thyroid cancer, renal cancer, liver cancer and leukemia.Optionally, the proliferative disorder is metastatic.

In therapeutic applications, compositions are administered to a subjectsuffering from a proliferative disease or disorder (e.g., cancer) in a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. A “patient” or “subject” includes bothhumans and other animals, particularly mammals. Thus the methods areapplicable to both human therapy and veterinary applications.

Optionally, the provided methods include administering to the subjectone or more additional therapeutic agents. Thus, the provided methodscan be combined with other cancer therapies, radiation therapy, hormonetherapy, or chemotherapy. The combined administrations contemplatescoadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities. Combinations of agentsor compositions can be administered either concomitantly (e.g., as amixture), separately but simultaneously (e.g., via separate intravenouslines) or sequentially (e.g., one agent is administered first followedby administration of the second agent). Thus, the term combination isused to refer to concomitant, simultaneous or sequential administrationof two or more agents or compositions.

According to the methods provided herein, the subject is administered aneffective amount of one or more of the agents provided herein. The termseffective amount and effective dosage are used interchangeably. The termeffective amount is defined as any amount necessary to produce a desiredphysiologic response (e.g., killing of a cancer cell). The dosages,however, may be varied depending upon the requirements of the subject,the severity of the condition being treated, and the compound beingemployed. For example, dosages can be empirically determined consideringthe type and stage of cancer diagnosed in a particular subject. The doseadministered to a subject, in the context of the provided methods shouldbe sufficient to affect a beneficial therapeutic response in the patientover time. Determination of the proper dosage for a particular situationis within the skill of the practitioner. Thus, effective amounts andschedules for administering the agent may be determined empirically byone skilled in the art. The dosage ranges for administration are thoselarge enough to produce the desired effect in which one or more symptomsof the disease or disorder are affected (e.g., reduced or delayed). Thedosage should not be so large as to cause substantial adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sex,type of disease, the extent of the disease or disorder, route ofadministration, or whether other drugs are included in the regimen, andcan be determined by one of skill in the art. The dosage can be adjustedby the individual physician in the event of any contraindications.Dosages can vary and can be administered in one or more doseadministrations daily, for one or several days. Guidance can be found inthe literature for appropriate dosages for given classes ofpharmaceutical products.

As used herein the terms treatment, treat, or treating refers to amethod of reducing the effects of one or more symptoms of a disease orcondition characterized by expression of the protease or symptom of thedisease or condition characterized by expression of the protease. Thusin the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of anestablished disease, condition, or symptom of the disease or condition.For example, a method for treating a disease is considered to be atreatment if there is a 10% reduction in one or more symptoms of thedisease in a subject as compared to a control. Thus the reduction can bea 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentreduction in between 10% and 100% as compared to native or controllevels. It is understood that treatment does not necessarily refer to acure or complete ablation of the disease, condition, or symptoms of thedisease or condition. Further, as used herein, references to decreasing,reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater as compared to a control level and suchterms can include but do not necessarily include complete elimination.

Kits

Provided herein are kits for screening for compounds that bind orinhibit SENP1. The kits include a composition comprising an SENP1polypeptide. Optionally, the composition is an aqueous solution.Optionally, the SENP1 polypeptide comprises SEQ ID NO:1, 2, 3, 4, 5, 6,or 7. Optionally, the SENP1 polypeptide comprises amino acid residues440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 numberedrelative to SEQ ID NO:1. Optionally, the aqueous composition comprisingan SENP1 polypeptide is at a pH from about 6.0 to about 7.5. Optionally,the pH is about 6.8. Optionally, the compositions can further include,for example, buffering agents, reducing agents, bases and combinationsthereof. Optionally, the compositions can include sodium phosphate, D₂O,sodium azide, dithiothreitol or combinations thereof. By way of example,the sodium phosphate can be present at about 20 mM. Optionally, theSENP1 polypeptide is bound to a SUMO protein thereby forming aSENP1-SUMO complex. Optionally, the SENP1 polypeptide or SENP1-SUMOcomplex is bound to a compound thereby forming a SENP1-compound complexor SENP1-SUMO-compound complex. Optionally, the SUMO protein is atruncated SUMO protein. In some embodiments, the kit comprises acontainer including a SENP1 polypeptide or SENP1-SUMO complex and,optionally, a second container including a SENP1-compound complex orSENP-SUMO-compound complex.

Further provided are kits including an inhibitor of SENP1. Optionally,the kit comprises one or more doses of an effective amount of acomposition comprising a SENP1 inhibitor. Optionally, the composition ispresent in a container (e.g., vial or packet). Optionally, the kitfurther includes one or more additional therapeutic agents. Optionally,the therapeutic agent is a chemotherapeutic agent. Optionally, the kitcomprises a means of administering the composition, such as, forexample, a syringe, needle, tubing, catheter, patch, and the like. Thekit may also comprise formulations and/or materials requiringsterilization and/or dilution prior to use.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the claims.

EXAMPLE Example 1. Identification and Characterization of a SENPInhibitors

Enzymes called SENPs catalyze both the maturation of smallubiquitin-like modifier (SUMO) precursors and removal of SUMOmodifications, which regulate essential cellular functions such as cellcycle progression, DNA damage response and intracellular trafficking.Some members, such as SENP1, are potential targets for developing cancertherapeutics. A search for small molecule inhibitors of SENPs wascarried out using in-silico screening in conjunction with biochemicalassays, and a new chemotype of small molecule inhibitors thatnon-covalently inhibit SENPs was identified. The inhibitors confer thenon-competitive inhibitory mechanism, as shown by nuclear magneticresonance (NMR) and quantitative enzyme kinetic analysis. The NMR dataalso provided evidence for substrate-assisted inhibitor binding, whichindicates the need for caution in using artificial substrates forcompound screening, as the inhibitory effects could be significantlydifferent from using the physiological substrates.

In this study, it was purported to identify small molecule inhibitors ofSENPs through in-silico screening in conjunction with enzyme kinetic,nuclear magnetic resonance (NMR) and cellular analyses. In silicoscreening was performed using Protein Data Bank (PDB) accession codes2IYC and 2IY1 and by considering hydrogen bonding and hydrophobicinteractions between the C-terminus of full-length SUMO-1 and SENP1. TheGLIDE program (Friesner et al., Journal of Medicinal Chemistry47:1739-1749 (2004)) was used to search the 250,000 compound libraryprovided by the Developmental Therapeutics Program (DTP) of the NationalCancer Institute, using the E-model scoring function of Cvdw, which isthe sum of the van der Waals (Evdw) and electrostatic interaction energyterms (Eelec). Among the top hits, the dominant scaffolds werepeptidomimetics and compounds that contained 2-fold symmetry. Fortycompounds (100 μM) representing the dominant scaffolds were tested fortheir inhibitory effects on SENP1 and SENP2 for maturation of SUMO-1 andSUMO-2 precursors. The most potent compounds contained sulfonyl-benzenegroups. Additional analogues of this group were obtained from DTP, andNSC5068, hereafter referred to as SPI-01 (SUMO protease inhibitor), wasfound to have the highest potency (Table 1). Available analogs of SPI-01were obtained from DTP. Five compounds in this group (Table 1, SPI-06 toSPI-10) are “half” of the other compounds (Table 1, SPI-01 to SPI-05)and allowed the exploration of the activity requirements of the two-foldsymmetric structure of SPI-01 to SPI-05. The inhibitory activity ofthese compounds on SENP1 and SENP2 was characterized using substratesthat contained precursor SUMO-1 or SUMO-2 (S) flanked by yellowfluorescent protein (Y) at the N-terminus and enhanced cyan fluorescentprotein (E) at the C-terminus (YSE) (Tatham and Hay, Methods Mol. Biol.497:253-268 (2009)). Although the cleavage of the substrates can bedetected by fluorescence resonance energy transfer (FRET), FRET couldnot be used because many of these compounds interfere with the FRETsignal. Therefore, a gel-based assay was used to determine theinhibitory effects of all compounds on SENP1 and 2 (representative datashown in FIGS. 1 and 2), and the gel bands were quantified to determinethe half maximum inhibitory concentrations (IC₅₀) (Table 1). Theinhibitory effects of the compounds on the endopeptidase activities werenot only enzyme-dependent, but also substrate-dependent. ForSENP1-mediated cleavage of SUMO-1 precursor, only four of the compounds(SPI-01 to SPI-04) had half maximal inhibitory concentrations (IC₅₀)below 60 μM. The inhibitors were more potent for inhibiting SENP2 thanSENP1 for cleavage of the SUMO-1 precursor. However, for cleavage of theSUMO-2 precursor, some compounds (i.e. SPI-01 and SPI-04) had similarpotency for inhibiting SENP1 and SENP2, while others (i.e. SPI-07 andSPI-10) were more potent for inhibiting SENP1 than SENP2 or vice versa(i.e. SPI-06 and SPI-09) (Table 1). In addition to the differentialeffects on SENP1 and SENP2, SPI-01 had more than 10 fold less potencyfor inhibiting a de-ubiquitin enzyme isopeptidase T than inhibitingSENP2.

TABLE 1 Effect of inhibitors on inhibition of the maturation of SUMOprecursors by SENP1 and SENP2. Compounds IC₅₀ (μM)-SUMO1 IC₅₀ (μM)-SUMO2Structure Code^(†) NCI ID^(‡) SENP1 SENP2 SENP1 SENP2

SPI-01 NSC5068 32.8 ± 1.82 1.42 ± 3.0 1.88 ± 2.2 1.1 ± 5.8

SPI-02 NSC16224 26.5 ± 1.86 3.42 ± 1.6 2.08 ± 2.0 2.70 ± 2.1

SPI-03 NSC8676 20.27 ± 2.47 5.17 ± 1.32 1.86 ± 2.3 3.0 ± 2.0

SPI-04 NSC34933 11.2 ± 1.7 1.6 ± 2.5 2.32 ± 2.6 2.15 ± 2.28

SPI-05 NSC5067 >60 19.7 ± 1.47 7.5 ± 1.6 4.6 ± 1.65

SPI-06 NSC70551 >60 3.62 ± 1.98 4.32 ± 2.2 10.7 ± 1.6

SPI-07 NSC58046 >60 >60 17.54 ± 4.9 28.06 ± 9.2

SPI-08 NSC22940 >60 4.1 ± 3.0 >60 41.06 ± 5.2

SPI-09 NSC42164 >60 23.6 ± 1.6 >60 26.6 ± 2.5

SPI-10 NSC45551 >60 34.21 ± 1.9 11.1 ± 3.7 36.44 ± 5.7 ^(†)Designationfor our library of SUMO-protease inhibitors (SPI) ^(‡)Designated by theNational Cancer Institute

To determine whether other SENPs can be inhibited by this family ofinhibitors, a distant SENP member, SENP7, was tested in parallel withSENP1 and SENP2 using a pentapeptide substrate that contained theGly-Gly motif and luciferin, known as DUB-Glo (Promega, Madison, Wis.).Cleavage of luciferin by a SENP can be detected by a coupledbioluminescent assay using luciferase. The bioluminescent reporter waschosen instead of a fluorescent reporter to avoid interference by thecompounds during detection. In addition, because SENP7 has differentphysiological substrates than SENP1 and SENP2 (Kolli et al., BiochemicalJournal 430:335-344 (2010); and Shen et al., EMBO Rep. 13(4):339-46(2012)), an advantage of DUB-Glo is that it can act as a commonsubstrate for all SENPs, which enabled us to rule out substrate-specificeffects. The dose-dependent inhibition of each SENP by the inhibitorswas determined (FIGS. 3A-3C), as was the IC₅₀ for inhibition of SENP1, 2and 7 of all the compounds (Table 2). Most compounds had more similarinhibitory effects on SENP1 and SENP2 than on SENP7, consistent withtheir amino acid sequence similarities. In addition, the compounds weremore potent for inhibiting SENP1 when DUB-Glo was used as a substratethan when SUMO-1 precursor was used (Tables 1 and 2). To rule out thepossibility that these compounds used a promiscuous mechanism, thecompounds were also tested in SUMOylation and ubiquitination reactions,which also depend on enzymes containing catalytic Cys residues. Thecompounds were noninhibitory in these assays. Furthermore, comparison ofthe DUB-Glo and the SUMO maturation assays revealed that the effect ofSENP inhibitors could be highly substrate-specific.

TABLE 2 Inhibitory effect on SENP enzymatic activity using abioluminescent peptide substrate SENP1 SENP2 SENP7 Structure Code^(†)NCI ID^(‡) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM)

SPI-01 NSC5068 5.9 ± 1.4 2.9 ± 1.6 3.5 ± 1.5

SPI-02 NSC 16224 2.1 ± 1.9 2.0 ± 2.0 2.7 ± 1.8

SPI-03 NSC 8676 3.8 ± 1.5 2.4 ± 1.8 4.8 ± 1.4

SPI-04 NSC 34933 2.4 ± 1.8 2.3 ± 1.8 3.4 ± 1.5

SPI-05 NSC 5067 13.3 ± 1.3 8.5 ± 1.3 4.6 ± 1.5

SPI-06 NSC 70551 3.9 ± 1.4 3.7 ± 1.4 4.7 ± 1.7

SPI-07 NSC 58046 >>60 >>60 1.9 ± 2.2

SPI-08 NSC 22940 22.2 ± 1.5 17.2 ± 1.5 2.8 ± 1.6

SPI-09 NSC 42164 >60 6.8 ± 1.3 1.9 ± 2.1

SPI-10 NSC 45551 2.4 ± 1.8 2.5 ± 1.7 2.0 ± 2.0 ^(†)Designation for ourlibrary of SUMO-protease inhibitors (SPI) ^(‡)Designated by the NationalCancer Institute

The abilities of representative inhibitors were then tested to inhibitSENP in cells. HeLa cells were treated with increasing concentrations ofSPI-01 for 48 hours, after which SUMOylated proteins were detected inthe cells by Western blots. SUMO-2/3 conjugates accumulated in cells andthis accumulation correlated with inhibitor concentration, particularlyat high molecular weights (FIG. 4). This result suggests that SPI-01inhibits the isopeptidase activities of SENPs, particularly SENP6 andSENP7, which are required for SUMO chain editing. It was observed thatless significant effects on the accumulation of SUMO-1 conjugates,possibly because most SENPs cleave SUMO-2/3-conjugates. It is known thatheat shock triggers a dramatic increase in global SUMO-2/3 conjugationsand that during recovery, the SUMOylated proteins are removed, at leastin part, due to the deSUMOylation activity of SENP1 (Nefkens et al., J.Cell Sci. 116:513-524 (2003)). To further confirm that the inhibitorsinhibited deSUMOylase activities, HeLa cells were treated with SPI-01and SPI-02 for 2 hours at 37° C. Then, SPI-treated or untreated controlHeLa cells was transferred to 42° C. for 30 minutes, followed byrecovery for 4 hours at 37° C. before processing for detection of globalSUMO-2/3 levels. The inhibitor-treated cells had considerably higherlevels of SUMOylated proteins than did the corresponding controls thatdid not receive heat shock or the mock-treated cells after the recoveryperiod (FIG. 5). Thus, the results of the heat-shock experiments furtherconfirmed that the SPI compounds had inhibitory effects on SENPs incells.

NMR chemical shift perturbation (CSP) analysis was used to investigatewhether this family of inhibitors binds the enzyme or theenzyme-substrate complex. CSP experiments were conducted using a¹⁵N-labeled C603 S mutant of the human SENP1 catalytic domain(SENP1-C603S, for which NMR chemical shift assignments have beenobtained and deposited in the Biological Magnetic Resonance Bank (BMRB)with accession number 19083). Although the SENP1-C603S mutant iscatalytically inactive (Xu et al., Biochem. J. 398:345-52 (2006)), itretains binding activity for the precursor or mature SUMO paralogs orSUMOylated substrates (Shen et al., Nat. Struct. Mol. Biol. 13:1069-1077(2006)). It was observed that SPI-01 caused modest backbone amide CSPfor a subset of SENP1-C603S residues. Of note, specific CSPs wereobserved at the canonical cysteine-protease catalytic triad residues(D550, H533, and C603), the proposed dynamic channel of conserved W465and W534, and at several other residues located at or adjacent to theSENP catalytic center (W465, L466, G531, H533, W534, C535, M552, G554and Q596) with only one residue located distal to this surface (E469)(FIG. 2). Interestingly, M552, G554, and Q596 are clustered at the SENP1surface that contacts the C-terminal tail of SUMO-1. Supporting theimportance of this surface in SENP catalytic activity, non-conservativepoint mutations of Q596 in SENP1 or the equivalent residue to SENP1 M552in SENP2 (M497) perturb SUMO processing and deconjugation (Reverter andLima, Nat. Struct. Mol. Biol. 13; 1060-8 (2006); and Shen et al.,Biochem. J. 397:279-288 (2006)). Residue E469 is positioned toward thebinding surface for the structured region of SUMO-1, and its CSP may bedue to an alternative interaction with the compound or long-rangeeffects. These results indicate that SPI-01 binds the surface adjacentto the catalytic center that contacts the C-terminal portion of the SUMOprecursors. The residues that showed CSP are highly conserved betweenSENP1 and SENP2, suggesting that SPI-01 can interact with the equivalentsurface on SENP2.

The binding of SPI-01 to the enzyme-substrate complex was investigated.CSP analysis was carried out on the 40 kDa complex of ¹⁵N-labeled fulllength precursor SUMO-1-GGHSTV (SUMO-1-FL) with unlabeled SENP1-C603S.An equimolar amount of SPI-01 was added to the 1:1 enzyme-substratecomplex. The only observed CSP on the ¹⁵N-labeled precursor SUMO-1-FLwas on the C-terminal residues S99 and V101 (FIGS. 7 and 8) (Song etal., PNAS 101:14373-8 (2004)). This result indicates that SPI-01 bindsthe enzyme-substrate complex at the interface between SENP and theC-terminal tails of precursor SUMO-FL. X-ray crystal structures showedthat the C-terminal tail of precursor SUMO sits in and projects out ofthe catalytic tunnel of SENPs (Shen et al., Nat. Struct. Mol. Biol.13:1069-77 (2006)). In the case of SENP1, the region that interacts withthe projected C-terminus is predominantly acidic and favors theC-terminus of SUMO-1, which is polar and positively charged, over thatof SUMO-2, whose C-terminus is mainly hydrophobic (Shen et al., Nat.Struct. Mol. Biol. 13:1069-77 (2006); and Shen et al., The BiochemicalJournal 397:279-88 (2006)). In addition, the more hydrophobic C-terminusof SUMO-2 may favor binding of aromatic inhibitors. These properties mayaccount for the more potent inhibition of processing of the SUMO-2precursor (Table 1).

To further investigate the inhibitory mechanism, enzyme kineticexperiments were conducted using the pentapeptide substrate DUB-Glo(FIG. 9). The data was fit to a mixed inhibition mechanism, as describedby the kinetic equation:

$v = \frac{V_{\max}\lbrack S\rbrack}{\left( {1 + \frac{\lbrack I\rbrack}{\alpha\; K_{i}}} \right)\left\lbrack {\frac{K_{m}\left( {1 + \frac{\lbrack I\rbrack}{K_{i}}} \right)}{1 + \frac{\lbrack I\rbrack}{\alpha\; K_{i}}} + \lbrack S\rbrack} \right\rbrack}$

in which the value of “α” indicates the mechanism of inhibition (Segel,Enzyme Kinetics John Wiley & Sons (1993)). For both SENP1 and SENP2, the“α” values indicated that the inhibitory mechanism is mainlynoncompetitive and suggests that the inhibitor binds to the enzyme andthe enzyme-substrate complex to inhibit chemical conversion. Thisfinding is consistent with the NMR binding analysis indicating that theinhibitor binds both the enzyme and the enzyme-substrate complex asdiscussed above.

In conclusion, this study has identified SENP inhibitors that do notcovalently modify the catalytic Cys residue. This study has alsoprovided the first mechanistic insights into how a small moleculeinhibitor of SENPs that does not covalently modify the catalytic Cys caninhibit the enzymes. The substrate-assisted inhibitor binding indicatesthe need for caution in designing high throughput screening assays thatuse fluorogenic or chemiluminescent artificial substrates, as theresults could be significantly different from using the physiologicalsubstrates. The substrate-dependent inhibitory effect suggests thepossibility of designing SENP inhibitors that are tuned forsubstrate-specificity.

Materials and Methods

Protein Purification. The catalytic domains of SENP1, 2, and 7 wereexpressed as His-tagged protein in E. coli (DE3) and purified usingnickel affinity chromatography (Namanja et al., The Journal ofBiological Chemistry 287:3231-3240 (2012)). The pET11 expressionplasmids for SENP1 and 2 contained a cDNA insert coding for thecatalytic domain of human SENP1-WT (419-644) and SENP2-WT (364-589). Theexpression plasmid for the SENP1 active site point mutant C603 S wasgenerated using the QuikChange mutagenesis kit (Agilent Technologies,San Diego, Calif.). The expression plasmid for the catalytic domain ofSENP7 has been described (Mikolajczyk et al., Journal of BiologicalChemistry 282:26217-26224 (2007)).

SUMO Cleavage Assays. SUMO cleavage assays were performed by incubatingSENPs with various concentrations of the inhibitor (0-60 μM) at roomtemperature for 10 min in assay buffer (50 mM Tris, pH 7.4, 100 mM NaCl,10 mM DTT). SENP concentrations were 32-50 nM when 50 μg/ml of the finalsubstrate YFP-SUMO-ECFP (YSE) fusion protein was added. The mixture wasincubated (37° C., 15 min), followed by SDS-PAGE and Coomassie stainingfor visualization. For cellular SENP inhibition experiments, HeLa cellscultured in DMEM plus 10% FBS, 100 units/ml penicillin, 100 mg/mlstreptomycin, and 0.2 M glutamine were treated for 48 hours with SPIcompounds. For heat shock experiment, HeLa cells were treated with SPIcompounds or mock treated (2 h, 37° C.), after which cells weretransferred to 42° C. for 30 min. After heat shock, the cells wereallowed to recover (4-5 hours) before being harvested and lysed.Proteins were separated by SDS-PAGE and immunoblotted to determineglobal SUMO-2/3 levels.

DUB-Glo Assay. The luciferase substrate assay (DUB-Glo, Promega,Madison, Wis.) was performed according to the manufacturer'sinstructions. Briefly, SENPs (final concentration 50-100 nM) in Trisbuffer (50 mM Tris, pH 8.0, 100 mM NaCl, 10 mM DTT) were pre-incubated(10 min, room temperature) with increasing concentrations of inhibitor(0-60 μM final concentration) followed by addition of the luciferasesubstrate. Luciferase output was recorded 30 min after addition of theluciferase substrate. Values are the averages of experiments performedin triplicate.

NMR Experiments. Samples used for NMR titration or chemical shiftperturbation analyses were ¹⁵N or ¹⁵N/¹³C-labeled; the titrant proteinor SPI-01 was not labeled. The ¹⁵N/¹³C SUMO-1-FL sample was used toextend the backbone assignments of mature SUMO-1 to the HSTV tail byusing 2D-¹⁵N-¹H-HSQC, 3D-HNCA, 3D-HNCOCA, and 3D-HNCACB. Additionally,comparison of ¹⁵N-¹H-HSQC between precursor and mature SUMO quicklyidentified the resonances of the HSTV tail. For SENP1 assignments, afull suite of triple-resonance NMR experiments were acquired on¹⁵N/¹³C/²H or ¹⁵N/13C samples: HNCA, HNCOCA, HNCACB, HNCOCACB, HNCO,HNCACO, and NOESY-HSQC. All samples were dissolved in the NMR buffer: 20mM sodium phosphate (pH 6.8), 10% D2O, 0.03% sodium azide and 10 mMd10-dithiothreitol. Purified perdeuterated SENP1 samples were unfoldedand refolded into NMR buffer.

For titration of SENP1-C603S with SPI-01, 270 μM ¹⁵N-labeled sample wastitrated with the inhibitor that was prepared by diluting a 10 mM stockin 100% DMSO-d₆ to a concentration of 1.7 mM in the NMR buffer. The 2D¹H-¹⁵N-HSQC spectra of SENP1 were recorded at each incremental additionof 5 μl of SPI-01 into 250 μl of SENP1. The chemical shift perturbation(CSP) analysis compared the spectra of SENP1 in the absence or thepresence of equimolar SPI-01. A separate DMSO control titration wasperformed to account for DMSO-induced CSP. NMR resonance assignments forSUMO samples at 35° C. were transferred from those obtained at 25° C. byspectral acquisition at 2.5° C. incremental increases. All data wereacquired on a 600 MHz Bruker Avance NMR spectrometer equipped with a TXICryoprobe.

TABLE 3 Free SENP1 NMR Chemical Shifts Values. Chemical Shift AmbiguityIndex Value Definitions The values other than 1 are used for those atomswith different chemical shifts that cannot be assigned to stereospecificatoms or to specific residues or chains. Index Value Definition 1 Unique(including isolated methyl protons germinal atoms, and geminal methylgroups with identical chemical shifts (e.g. ILE HD11, HD12, HD13protons) 2 Ambiguity of geminal atoms or geminal methyl proton groups(e.g. ASP HB2 and HB3 protons, LEU CD1 and CD2 carbons, or LEU HD11,HD12, HD13 and HD21, HD22, HD23 methyl protons) 3 Aromatic atoms onopposite sides of symmetrical rings (e.g. TYR HE1 and HE2 protons) 4Intraresidue ambiguities (e.g. LYS HG and HD protons or TRP HZ2 and HZ3protons) 5 Interresidue ambiguities (LYS 12 vs. LYS 27) 6 Intermolecularambiguities (e.g. ASP 31 CA in monomer 1 and ASP 31 CA in monomer 2 ofan asymmetrical homodimer, duplex DNA assignments, or other assignmentsthat may apply to atoms in one or more molecule in the molecularassembly) 9 Ambiguous, specific ambiguity not defined Chemical AtomResidue Amino Atom Atom Iso- shift Unique- number number acid contexttype type (ppm)* ness 1 419 E CA C 13 56.635 1 2 419 E CB C 13 29.326 13 419 E CO C 13 175.803 1 4 419 E H H 1 8.056 1 5 419 E N N 15 120.257 16 420 F CA C 13 54.951 1 7 420 F CB C 13 37.882 1 8 420 F CO C 13173.207 1 9 420 F H H 1 8.035 1 10 420 F N N 15 118.648 1 11 422 E CA C13 56.498 1 12 422 E CB C 13 29.476 1 13 422 E CO C 13 176.111 1 14 422E H H 1 8.637 1 15 422 E N N 15 124.065 1 16 423 I CA C 13 60.725 1 17423 I CB C 13 35.427 1 18 423 I CO C 13 176.659 1 19 423 I H H 1 8.522 120 423 I N N 15 122.041 1 21 424 T CB C 13 70.292 1 22 424 T CO C 13174.725 1 23 424 T H H 1 7.633 1 24 424 T N N 15 121.188 1 25 425 E CA C13 59.696 1 26 425 E CB C 13 28.462 1 27 425 E H H 1 8.913 1 28 425 E NN 15 120.9 1 29 426 E CA C 13 59.444 1 30 426 E CO C 13 179.787 1 31 426E H H 1 8.419 1 32 426 E N N 15 118.024 1 33 427 M CB C 13 33.371 1 34427 M CO C 13 177.912 1 35 427 M H H 1 7.366 1 36 427 M N N 15 119.301 137 428 E CB C 13 28.41 1 38 428 E CO C 13 178.828 1 39 428 E H H 1 8.6051 40 428 E N N 15 118.58 1 41 429 K CA C 13 59.488 1 42 429 K CB C 1331.407 1 43 429 K CO C 13 178.978 1 44 429 K H H 1 7.858 1 45 429 K N N15 118.101 1 46 430 E CB C 13 29.584 1 47 430 E CO C 13 179.111 1 48 430E H H 1 7.356 1 49 430 E N N 15 119.087 1 50 431 I CA C 13 64.567 1 51431 I CB C 13 38.123 1 52 431 I CO C 13 176.796 1 53 431 I H H 1 8.075 154 431 I N N 15 119.774 1 55 432 K CA C 13 59.195 1 56 432 K CB C 1331.197 1 57 432 K CO C 13 180.185 1 58 432 K H H 1 8.32 1 59 432 K N N15 116.686 1 60 433 N CA C 13 55.822 1 61 433 N CB C 13 37.95 1 62 433 NCO C 13 178.466 1 63 433 N H H 1 7.635 1 64 433 N N N 15 114.913 1 65434 V CA C 13 64.189 1 66 434 V CB C 13 30.396 1 67 434 V CG1 C 1322.475 1 68 434 V CG2 C 13 21.674 1 69 434 V CO C 13 176.412 1 70 434 VH H 1 7.548 1 71 434 V HG1 H 1 0.724 1 72 434 V HG2 H 1 0.725 1 73 434 VN N 15 114.74 1 74 435 F CA C 13 55.321 1 75 435 F CB C 13 37.701 1 76435 F CO C 13 177.18 1 77 435 F H H 1 7.344 1 78 435 F N N 15 117.562 179 436 R CA C 13 56.384 1 80 436 R CB C 13 30.009 1 81 436 R CO C 13176.099 1 82 436 R H H 1 7.225 1 83 436 R N N 15 118.73 1 84 437 N CA C13 53.605 1 85 437 N CB C 13 38.193 1 86 437 N H H 1 8.252 1 87 437 N NN 15 119.685 1 88 438 G CA C 13 44.807 1 89 438 G CO C 13 172.725 1 90438 G H H 1 8.08 1 91 438 G N N 15 109.481 1 92 439 N CA C 13 52.847 193 439 N CB C 13 37.175 1 94 439 N H H 1 8.737 1 95 439 N N N 15 120.4431 96 440 Q CA C 13 58.04 1 97 440 Q CB C 13 28.495 1 98 440 Q CO C 13175.605 1 99 440 Q H H 1 9.022 1 100 440 Q N N 15 125.59 1 101 441 D CAC 13 53.428 1 102 441 D CB C 13 40.409 1 103 441 D CO C 13 175.45 1 104441 D H H 1 7.969 1 105 441 D N N 15 114.845 1 106 442 E CA C 13 56.5011 107 442 E CB C 13 30.044 1 108 442 E CO C 13 175.905 1 109 442 E H H 17.143 1 110 442 E N N 15 121.501 1 111 443 V CA C 13 64.02 1 112 443 VCB C 13 31.155 1 113 443 V CG1 C 13 21.487 1 114 443 V CG2 C 13 21.844 1115 443 V H H 1 8.65 1 116 443 V HG1 H 1 0.721 1 117 443 V HG2 H 1 0.8821 118 443 V N N 15 127.026 1 119 444 L CA C 13 54.051 1 120 444 L CB C13 43.293 1 121 444 L CD1 C 13 27.029 1 122 444 L CD2 C 13 21.807 1 123444 L CO C 13 176.79 1 124 444 L H H 1 9.012 1 125 444 L HD1 H 1 0.59 1126 444 L HD2 H 1 0.597 1 127 444 L N N 15 127.296 1 128 445 S CA C 1357.432 1 129 445 S CB C 13 64.093 1 130 445 S CO C 13 172 1 131 445 S HH 1 7.412 1 132 445 S N N 15 111.726 1 133 446 E CA C 13 55.317 1 134446 E CB C 13 31.965 1 135 446 E CO C 13 174.257 1 136 446 E H H 1 7.9331 137 446 E N N 15 125.063 1 138 447 A CA C 13 51.875 1 139 447 A CB C13 18.979 1 140 447 A CO C 13 176.087 1 141 447 A H H 1 8.286 1 142 447A N N 15 124.213 1 143 448 F CA C 13 56.316 1 144 448 F CB C 13 36.029 1145 448 F CO C 13 175.788 1 146 448 F H H 1 8.61 1 147 448 F N N 15115.367 1 148 449 R CA C 13 57.675 1 149 449 R CB C 13 26.229 1 150 449R CO C 13 175.491 1 151 449 R H H 1 8.484 1 152 449 R N N 15 110.844 1153 450 L CA C 13 53.763 1 154 450 L CB C 13 44.035 1 155 450 L CD1 C 1325.813 1 156 450 L CD2 C 13 22.437 1 157 450 L CO C 13 176.645 1 158 450L H H 1 8.389 1 159 450 L HD1 H 1 0.89 1 160 450 L HD2 H 1 0.948 1 161450 L N N 15 121.623 1 162 451 T CA C 13 60.834 1 163 451 T CB C 1371.178 1 164 451 T CO C 13 173.407 1 165 451 T H H 1 8.315 1 166 451 T NN 15 113.296 1 167 452 I CA C 13 56.522 1 168 452 I CB C 13 36.082 1 169452 I CO C 13 176.082 1 170 452 I H H 1 8.521 1 171 452 I N N 15 124.1731 172 453 T CA C 13 59.709 1 173 453 T CB C 13 72.939 1 174 453 T H H 19.811 1 175 453 T N N 15 119.807 1 176 454 R CA C 13 60.137 1 177 454 RCB C 13 28.989 1 178 454 R CO C 13 177.392 1 179 454 R H H 1 8.211 1 180454 R N N 15 122.061 1 181 455 K CA C 13 59.289 1 182 455 K CB C 1331.114 1 183 455 K CO C 13 178.628 1 184 455 K H H 1 8.504 1 185 455 K NN 15 119.122 1 186 456 D CA C 13 57.369 1 187 456 D CB C 13 40.809 1 188456 D H H 1 7.271 1 189 456 D N N 15 117.779 1 190 457 I CA C 13 62.3921 191 457 I CB C 13 37.06 1 192 457 I H H 1 8.159 1 193 457 I N N 15121.588 1 194 458 Q CA C 13 57.804 1 195 458 Q CB C 13 26.567 1 196 458Q CO C 13 178.732 1 197 458 Q H H 1 7.923 1 198 458 Q N N 15 117.897 1199 459 T CA C 13 65.051 1 200 459 T CB C 13 67.395 1 201 459 T H H 17.897 1 202 459 T N N 15 113.263 1 203 460 L CA C 13 54.923 1 204 460 LCB C 13 41.723 1 205 460 L CD1 C 13 25.968 1 206 460 L CD2 C 13 25.889 1207 460 L CO C 13 179.644 1 208 460 L H H 1 7.253 1 209 460 L HD1 H 10.82 1 210 460 L HD2 H 1 0.925 1 211 460 L N N 15 115.083 1 212 461 N CAC 13 51.888 1 213 461 N CB C 13 37.194 1 214 461 N H H 1 7.421 1 215 461N N N 15 119.845 1 216 462 H CA C 13 57.014 1 217 462 H CB C 13 28.992 1218 462 H H H 1 7.773 1 219 462 H N N 15 119.821 1 220 465 W CA C 1356.901 1 221 465 W CB C 13 27.801 1 222 465 W H H 1 8.319 1 223 465 WHE1 H 1 10.206 1 224 465 W N N 15 120.321 1 225 465 W NE1 N 15 130.435 1226 466 L CA C 13 57.74 1 227 466 L CB C 13 41.916 1 228 466 L CD1 C 1325.446 1 229 466 L CD2 C 13 23.298 1 230 466 L H H 1 7.644 1 231 466 LHD1 H 1 0.634 1 232 466 L HD2 H 1 0.563 1 233 466 L N N 15 125.508 1 234467 N CA C 13 50.295 1 235 467 N CB C 13 39.556 1 236 467 N CO C 13174.619 1 237 467 N H H 1 7.164 1 238 467 N N N 15 116.901 1 239 468 DCA C 13 57.481 1 240 468 D CB C 13 40.531 1 241 468 D H H 1 8.246 1 242468 D N N 15 115.434 1 243 469 E CA C 13 60.578 1 244 469 E CB C 1327.414 1 245 469 E CO C 13 179.948 1 246 469 E H H 1 8.991 1 247 469 E NN 15 119.089 1 248 470 I CA C 13 61.231 1 249 470 I CB C 13 34.744 1 250470 I CO C 13 177.04 1 251 470 I H H 1 7.753 1 252 470 I N N 15 117.8451 253 471 I CA C 13 64.903 1 254 471 I H H 1 6.974 1 255 471 I N N 15117.941 1 256 472 N CA C 13 56.07 1 257 472 N CB C 13 37.624 1 258 472 NCO C 13 178.288 1 259 472 N H H 1 9.03 1 260 472 N N N 15 115.254 1 261473 F CA C 13 62.584 1 262 473 F CB C 13 39.667 1 263 473 F CO C 13177.436 1 264 473 F H H 1 8.304 1 265 473 F N N 15 123.63 1 266 474 Y CAC 13 62.784 1 267 474 Y CB C 13 38.335 1 268 474 Y CO C 13 178.151 1 269474 Y H H 1 8.774 1 270 474 Y N N 15 120.467 1 271 475 M CA C 13 57.3251 272 475 M CB C 13 31.041 1 273 475 M CO C 13 179.367 1 274 475 M H H 18.709 1 275 475 M N N 15 115.346 1 276 476 N CA C 13 56.292 1 277 476 NCB C 13 37.912 1 278 476 N CO C 13 177.604 1 279 476 N H H 1 7.371 1 280476 N N N 15 117.074 1 281 477 M CA C 13 59.952 1 282 477 M CB C 1331.504 1 283 477 M CO C 13 179.545 1 284 477 M H H 1 7.664 1 285 477 M NN 15 121.465 1 286 478 L CA C 13 57.471 1 287 478 L CB C 13 39.79 1 288478 L CD1 C 13 27.34 1 289 478 L CD2 C 13 22.112 1 290 478 L CO C 13180.857 1 291 478 L H H 1 7.767 1 292 478 L HD1 H 1 0.658 1 293 478 LHD2 H 1 0.411 1 294 478 L N N 15 119.925 1 295 479 M CA C 13 59.413 1296 479 M CB C 13 32.433 1 297 479 M CO C 13 179.134 1 298 479 M H H 17.603 1 299 479 M N N 15 118.957 1 300 480 E CA C 13 59.225 1 301 480 ECB C 13 28.37 1 302 480 E H H 1 8.059 1 303 480 E N N 15 122.932 1 304481 R CA C 13 58.251 1 305 481 R CB C 13 28.749 1 306 481 R CO C 13176.421 1 307 481 R H H 1 7.917 1 308 481 R N N 15 120.662 1 309 482 SCA C 13 60.255 1 310 482 S CB C 13 63.102 1 311 482 S CO C 13 172.394 1312 482 S H H 1 7.201 1 313 482 S N N 15 113.273 1 314 483 K CA C 1356.793 1 315 483 K CB C 13 31.687 1 316 483 K CO C 13 178.176 1 317 483K H H 1 6.968 1 318 483 K N N 15 118.755 1 319 484 E CB C 13 29.049 1320 484 E CO C 13 176.653 1 321 484 E H H 1 8.114 1 322 484 E N N 15121.011 1 323 485 K CA C 13 57.55 1 324 485 K CB C 13 31.154 1 325 485 KCO C 13 177.924 1 326 485 K H H 1 8.263 1 327 485 K N N 15 121.725 1 328486 G CA C 13 44.731 1 329 486 G CO C 13 173.993 1 330 486 G H H 1 8.7381 331 486 G N N 15 111.446 1 332 487 L CA C 13 52.224 1 333 487 L CB C13 40.075 1 334 487 L CD1 C 13 25.797 1 335 487 L CD2 C 13 23.228 1 336487 L CO C 13 174.966 1 337 487 L H H 1 7.357 1 338 487 L HD1 H 1 0.7781 339 487 L HD2 H 1 0.829 1 340 487 L N N 15 121.648 1 341 489 S CA C 1357.732 1 342 489 S CB C 13 63.976 1 343 489 S CO C 13 175.307 1 344 489S H H 1 9.146 1 345 489 S N N 15 117.954 1 346 490 V CA C 13 59.96 1 347490 V CB C 13 36.725 1 348 490 V CG1 C 13 21.034 1 349 490 V CG2 C 1323.035 1 350 490 V CO C 13 175.445 1 351 490 V H H 1 7.378 1 352 490 VHG1 H 1 0.555 1 353 490 V HG2 H 1 0.885 1 354 490 V N N 15 118.616 1 355491 H CA C 13 56.457 1 356 491 H CB C 13 33.175 1 357 491 H CO C 13172.689 1 358 491 H H H 1 8.824 1 359 491 H N N 15 124.16 1 360 492 A CAC 13 48.933 1 361 492 A CB C 13 20.637 1 362 492 A CO C 13 175.149 1 363492 A H H 1 7.475 1 364 492 A N N 15 129.587 1 365 493 F CA C 13 57.4431 366 493 F CB C 13 40.032 1 367 493 F H H 1 8.075 1 368 493 F N N 15120.292 1 369 494 N CA C 13 52.612 1 370 494 N CB C 13 39.014 1 371 494N CO C 13 177.042 1 372 494 N H H 1 8.614 1 373 494 N N N 15 116.324 1374 495 T CA C 13 65.108 1 375 495 T CB C 13 67.954 1 376 495 T H H 18.712 1 377 495 T N N 15 111.881 1 378 496 F CA C 13 57.589 1 379 496 FCB C 13 38.722 1 380 496 F CO C 13 176.791 1 381 496 F H H 1 8.441 1 382496 F N N 15 120.392 1 383 497 F CA C 13 61.468 1 384 497 F CB C 1338.442 1 385 497 F CO C 13 175.62 1 386 497 F H H 1 7.951 1 387 497 F NN 15 121.386 1 388 498 F CA C 13 62.45 1 389 498 F CB C 13 37.649 1 390498 F CO C 13 176.151 1 391 498 F H H 1 10.059 1 392 498 F N N 15120.473 1 393 499 T CA C 13 65.751 1 394 499 T CB C 13 68.656 1 395 499T H H 1 7.099 1 396 499 T N N 15 111.797 1 397 500 K CA C 13 58.082 1398 500 K CB C 13 30.293 1 399 500 K CO C 13 177.17 1 400 500 K H H 17.805 1 401 500 K N N 15 122.907 1 402 501 L CA C 13 56.922 1 403 501 LCB C 13 40.32 1 404 501 L CD1 C 13 21.344 1 405 501 L CD2 C 13 26.13 1406 501 L H H 1 8.04 1 407 501 L HD1 H 1 0.619 1 408 501 L HD2 H 1 0.2691 409 501 L N N 15 120.722 1 410 502 K CA C 13 58.359 1 411 502 K CB C13 31.117 1 412 502 K CO C 13 177.542 1 413 502 K H H 1 8.113 1 414 502K N N 15 117.113 1 415 503 T CA C 13 63.65 1 416 503 T CB C 13 69.641 1417 503 T CO C 13 175.362 1 418 503 T H H 1 7.521 1 419 503 T N N 15108.626 1 420 504 A CA C 13 51.681 1 421 504 A CB C 13 19.982 1 422 504A CO C 13 177.923 1 423 504 A H H 1 8.417 1 424 504 A N N 15 124.229 1425 505 G CA C 13 44.062 1 426 505 G CO C 13 173.703 1 427 505 G H H 17.404 1 428 505 G N N 15 108.216 1 429 506 Y CA C 13 61.372 1 430 506 YCB C 13 38.185 1 431 506 Y CO C 13 177.707 1 432 506 Y H H 1 8.506 1 433506 Y N N 15 118.015 1 434 507 Q CA C 13 58.073 1 435 507 Q CB C 1326.321 1 436 507 Q CO C 13 177.318 1 437 507 Q H H 1 8.677 1 438 507 Q NN 15 113.949 1 439 508 A CA C 13 53.059 1 440 508 A CB C 13 19.41 1 441508 A CO C 13 178.474 1 442 508 A H H 1 7.193 1 443 508 A N N 15 117.811 444 509 V CA C 13 59.584 1 445 509 V CB C 13 32.636 1 446 509 V CG1 C13 19.036 1 447 509 V CG2 C 13 20.077 1 448 509 V CO C 13 178.833 1 449509 V H H 1 6.99 1 450 509 V HG1 H 1 0.152 1 451 509 V HG2 H 1 0.505 1452 509 V N N 15 104.928 1 453 510 K CA C 13 59.235 1 454 510 K CB C 1330.396 1 455 510 K CO C 13 178.002 1 456 510 K H H 1 7.252 1 457 510 K NN 15 126.565 1 458 511 R CA C 13 56.969 1 459 511 R CB C 13 28.393 1 460511 R H H 1 8.593 1 461 511 R N N 15 116.236 1 462 512 W CA C 13 59.1541 463 512 W CB C 13 27.825 1 464 512 W CO C 13 178.179 1 465 512 W H H 18.477 1 466 512 W HE1 H 1 10.293 1 467 512 W N N 15 120.092 1 468 512 WNE1 N 15 129.338 1 469 513 T CA C 13 60 1 470 513 T CB C 13 65.562 1 471513 T CO C 13 174.181 1 472 513 T H H 1 7.356 1 473 513 T N N 15 105.8361 474 514 K CA C 13 59.285 1 475 514 K CB C 13 31.488 1 476 514 K CO C13 177.271 1 477 514 K H H 1 7.187 1 478 514 K N N 15 120.77 1 479 515 KCA C 13 55.075 1 480 515 K CB C 13 31.34 1 481 515 K CO C 13 175.55 1482 515 K H H 1 8.52 1 483 515 K N N 15 115.267 1 484 516 V CA C 1360.315 1 485 516 V CB C 13 34.794 1 486 516 V CG1 C 13 22.213 1 487 516V CG2 C 13 19.431 1 488 516 V CO C 13 173.373 1 489 516 V H H 1 7.346 1490 516 V HG1 H 1 1.035 1 491 516 V HG2 H 1 0.828 1 492 516 V N N 15118.521 1 493 517 D CA C 13 50.719 1 494 517 D CB C 13 39.298 1 495 517D CO C 13 178.171 1 496 517 D H H 1 8.502 1 497 517 D N N 15 124.325 1498 518 V CA C 13 64.12 1 499 518 V CB C 13 30.53 1 500 518 V CG1 C 1321.974 1 501 518 V CG2 C 13 17.74 1 502 518 V CO C 13 173.205 1 503 518V H H 1 8.909 1 504 518 V HG1 H 1 0.709 1 505 518 V HG2 H 1 0.246 1 506518 V N N 15 121.419 1 507 519 F CA C 13 57.9 1 508 519 F CB C 13 36.8721 509 519 F CO C 13 176.5 1 510 519 F H H 1 7.223 1 511 519 F N N 15110.893 1 512 520 S CB C 13 64.082 1 513 520 S CO C 13 173.635 1 514 520S H H 1 7.457 1 515 520 S N N 15 113.527 1 516 521 V CA C 13 58.421 1517 521 V CB C 13 33.049 1 518 521 V CG1 C 13 21.474 1 519 521 V CG2 C13 19.203 1 520 521 V CO C 13 174.363 1 521 521 V H H 1 6.675 1 522 521V HG1 H 1 0.677 1 523 521 V HG2 H 1 0.736 1 524 521 V N N 15 114.244 1525 522 D CA C 13 57.671 1 526 522 D CB C 13 42.241 1 527 522 D H H 18.177 1 528 522 D N N 15 120.102 1 529 523 I CA C 13 59.234 1 530 523 IH H 1 8.209 1 531 523 I N N 15 117.31 1 532 524 L CA C 13 51.912 1 533524 L CB C 13 42.117 1 534 524 L CD1 C 13 24.261 2 535 524 L CD2 C 1324.458 2 536 524 L H H 1 9.357 1 537 524 L HD1 H 1 0.826 2 538 524 L HD2H 1 0.873 2 539 524 L N N 15 121.905 1 540 525 L CA C 13 53.109 1 541525 L CB C 13 43.667 1 542 525 L CD1 C 13 27.473 1 543 525 L CD2 C 1323.613 1 544 525 L H H 1 8.708 1 545 525 L HD1 H 1 0.737 1 546 525 L HD2H 1 0.713 1 547 525 L N N 15 120.45 1 548 526 V CA C 13 59.564 1 549 526V CB C 13 32.337 1 550 526 V CG1 C 13 20.681 1 551 526 V CG2 C 13 19.4011 552 526 V H H 1 8.925 1 553 526 V HG1 H 1 −0.236 1 554 526 V HG2 H 10.488 1 555 526 V N N 15 120.847 1 556 528 I CA C 13 60.95 1 557 528 ICB C 13 39.801 1 558 528 I H H 1 8.737 1 559 528 I N N 15 125.023 1 560529 H CA C 13 50.979 1 561 529 H CB C 13 29.375 1 562 529 H CO C 13174.067 1 563 529 H H H 1 9.036 1 564 529 H N N 15 129.849 1 565 530 LCA C 13 52.799 1 566 530 L CB C 13 41.01 1 567 530 L CD1 C 13 25.841 1568 530 L CD2 C 13 23.767 1 569 530 L CO C 13 176.318 1 570 530 L H H 18.525 1 571 530 L HD1 H 1 0.874 1 572 530 L HD2 H 1 0.77 1 573 530 L N N15 130.501 1 574 531 G CA C 13 46.001 1 575 531 G CO C 13 174.79 1 576531 G H H 1 8.157 1 577 531 G N N 15 115.336 1 578 532 V CA C 13 61.0941 579 532 V CB C 13 30.784 1 580 532 V CG1 C 13 20.964 1 581 532 V CG2 C13 18.117 1 582 532 V CO C 13 175.461 1 583 532 V H H 1 8.198 1 584 532V HG1 H 1 0.532 1 585 532 V HG2 H 1 0.584 1 586 532 V N N 15 119.81 1587 533 H CA C 13 55.28 1 588 533 H CB C 13 32.996 1 589 533 H CO C 13174.498 1 590 533 H H H 1 7.803 1 591 533 H N N 15 121.771 1 592 534 WCA C 13 55.915 1 593 534 W CB C 13 32.49 1 594 534 W H H 1 6.407 1 595534 W HE1 H 1 9.377 1 596 534 W N N 15 125.3 1 597 534 W NE1 N 15128.192 1 598 535 C CA C 13 56.548 1 599 535 C CB C 13 30.615 1 600 535C CO C 13 171.965 1 601 535 C H H 1 9.461 1 602 535 C N N 15 117.22 1603 536 L CA C 13 54.008 1 604 536 L CB C 13 46.487 1 605 536 L CD1 C 1322.301 1 606 536 L CD2 C 13 26.282 1 607 536 L H H 1 7.905 1 608 536 LHD1 H 1 0.679 1 609 536 L HD2 H 1 0.597 1 610 536 L N N 15 120.825 1 611537 A CA C 13 49.576 1 612 537 A CB C 13 20.964 1 613 537 A H H 1 8.8351 614 537 A N N 15 126.773 1 615 538 V CA C 13 60.449 1 616 538 V CB C13 35.413 1 617 538 V CG1 C 13 21.698 1 618 538 V CG2 C 13 21.913 1 619538 V CO C 13 174.727 1 620 538 V H H 1 9.071 1 621 538 V HG1 H 1 0.87 1622 538 V HG2 H 1 0.809 1 623 538 V N N 15 119.546 1 624 539 V CA C 1360.873 1 625 539 V CB C 13 32.053 1 626 539 V CG1 C 13 20.502 1 627 539V CG2 C 13 19.475 1 628 539 V H H 1 9.402 1 629 539 V HG1 H 1 0.441 1630 539 V HG2 H 1 0.881 1 631 539 V N N 15 130.501 1 632 540 D CA C 1351.922 1 633 540 D CB C 13 41.816 1 634 540 D H H 1 8.954 1 635 540 D NN 15 126.546 1 636 541 F CA C 13 62.022 1 637 541 F CB C 13 39.187 1 638541 F H H 1 9.479 1 639 541 F N N 15 123.936 1 640 542 R CA C 13 57.3 1641 542 R CB C 13 28.607 1 642 542 R CO C 13 179.074 1 643 542 R H H 18.714 1 644 542 R N N 15 117.561 1 645 543 K CA C 13 54.808 1 646 543 KCB C 13 33.288 1 647 543 K CO C 13 175.221 1 648 543 K H H 1 6.749 1 649543 K N N 15 114.224 1 650 544 K CA C 13 55.562 1 651 544 K CB C 1327.641 1 652 544 K CO C 13 175.247 1 653 544 K H H 1 7.423 1 654 544 K NN 15 115.776 1 655 545 N CA C 13 51.023 1 656 545 N CB C 13 42.123 1 657545 N CO C 13 173.859 1 658 545 N H H 1 7.23 1 659 545 N N N 15 113.4981 660 546 I CA C 13 61.517 1 661 546 I H H 1 8.432 1 662 546 I N N 15120.288 1 663 547 T CA C 13 60.921 1 664 547 T CB C 13 70.493 1 665 547T H H 1 8.781 1 666 547 T N N 15 121.352 1 667 548 Y CA C 13 57.227 1668 548 Y CB C 13 40.796 1 669 548 Y H H 1 8.727 1 670 548 Y N N 15128.981 1 671 549 Y CB C 13 40.072 1 672 549 Y H H 1 9.079 1 673 549 Y NN 15 125.239 1 674 550 D CA C 13 52.549 1 675 550 D CB C 13 43.937 1 676550 D CO C 13 177.444 1 677 550 D H H 1 8.116 1 678 550 D N N 15 123.1691 679 551 S CA C 13 60.463 1 680 551 S CB C 13 62.962 1 681 551 S CO C13 174.422 1 682 551 S H H 1 9.519 1 683 551 S N N 15 122.969 1 684 552M CA C 13 54.901 1 685 552 M CB C 13 34.489 1 686 552 M CO C 13 178.9721 687 552 M H H 1 9.32 1 688 552 M N N 15 122.574 1 689 553 G CA C 1346.475 1 690 553 G CO C 13 175.385 1 691 553 G H H 1 7.89 1 692 553 G NN 15 109.507 1 693 554 G CA C 13 44.928 1 694 554 G CO C 13 171.658 1695 554 G H H 1 7.51 1 696 554 G N N 15 107.555 1 697 555 I CA C 1359.215 1 698 555 I CB C 13 37.955 1 699 555 I CO C 13 176.285 1 700 555I H H 1 8.05 1 701 555 I N N 15 118.138 1 702 556 N CA C 13 50.688 1 703556 N CB C 13 36.209 1 704 556 N H H 1 7.762 1 705 556 N N N 15 124.1061 706 557 N CA C 13 55.66 1 707 557 N CB C 13 37.225 1 708 557 N H H 18.339 1 709 557 N N N 15 121.312 1 710 558 E CA C 13 59.21 1 711 558 ECB C 13 28.223 1 712 558 E H H 1 8.531 1 713 558 E N N 15 120.721 1 714559 A CA C 13 55.072 1 715 559 A CB C 13 17.209 1 716 559 A CO C 13179.228 1 717 559 A H H 1 7.491 1 718 559 A N N 15 120.499 1 719 560 CCA C 13 61.861 1 720 560 C CB C 13 26.362 1 721 560 C CO C 13 176.152 1722 560 C H H 1 6.803 1 723 560 C N N 15 111.946 1 724 561 R CA C 1359.736 1 725 561 R CB C 13 29.122 1 726 561 R CO C 13 179.458 1 727 561R H H 1 8.077 1 728 561 R N N 15 120.238 1 729 562 I CA C 13 64.773 1730 562 I CB C 13 36.965 1 731 562 I CO C 13 179.283 1 732 562 I H H 18.583 1 733 562 I N N 15 120.588 1 734 563 L CA C 13 56.989 1 735 563 LCB C 13 41.139 1 736 563 L CD1 C 13 26.07 1 737 563 L CD2 C 13 22.794 1738 563 L CO C 13 177.668 1 739 563 L H H 1 7.58 1 740 563 L HD1 H 10.714 1 741 563 L HD2 H 1 0.791 1 742 563 L N N 15 120.645 1 743 564 LCA C 13 57.863 1 744 564 L CB C 13 40.586 1 745 564 L CD1 C 13 23.029 1746 564 L CD2 C 13 25.722 1 747 564 L H H 1 7.989 1 748 564 L HD1 H 10.503 1 749 564 L HD2 H 1 0.873 1 750 564 L N N 15 122.277 1 751 565 QCB C 13 27.023 1 752 565 Q CO C 13 178.554 1 753 565 Q H H 1 7.945 1 754565 Q N N 15 116.169 1 755 566 Y CA C 13 61.262 1 756 566 Y CB C 1336.89 1 757 566 Y H H 1 8.147 1 758 566 Y N N 15 121.414 1 759 567 L CAC 13 57.69 1 760 567 L CB C 13 39.693 1 761 567 L CD1 C 13 26.027 1 762567 L CD2 C 13 21.698 1 763 567 L H H 1 7.756 1 764 567 L HD1 H 1 0.2981 765 567 L HD2 H 1 0.574 1 766 567 L N N 15 118.927 1 767 568 K CA C 1359.666 1 768 568 K CB C 13 31.072 1 769 568 K CO C 13 180.159 1 770 568K H H 1 7.436 1 771 568 K N N 15 116.321 1 772 569 Q CA C 13 58.376 1773 569 Q CB C 13 26.837 1 774 569 Q CO C 13 178.121 1 775 569 Q H H 17.71 1 776 569 Q N N 15 119.281 1 777 570 E CA C 13 57.248 1 778 570 ECB C 13 27.85 1 779 570 E CO C 13 178.511 1 780 570 E H H 1 8.874 1 781570 E N N 15 123.757 1 782 571 S CA C 13 61.824 1 783 571 S CB C 1363.591 1 784 571 S H H 1 8.112 1 785 571 S N N 15 113.072 1 786 572 I CAC 13 64.046 1 787 572 I CB C 13 36.762 1 788 572 I CO C 13 178.898 1 789572 I H H 1 7.085 1 790 572 I N N 15 120.025 1 791 573 D CA C 13 58.5891 792 573 D CB C 13 45.295 1 793 573 D H H 1 8.267 1 794 573 D N N 15119.596 1 795 574 K CA C 13 55.261 1 796 574 K H H 1 8.537 1 797 574 K NN 15 110.111 1 798 575 K CA C 13 53.6 1 799 575 K H H 1 7.814 1 800 575K N N 15 114.921 1 801 580 D CA C 13 53.108 1 802 580 D CO C 13 175.9741 803 580 D H H 1 8.008 1 804 580 D N N 15 128.24 1 805 581 T CA C 1361.607 1 806 581 T CB C 13 67.992 1 807 581 T CO C 13 176.37 1 808 581 TH H 1 8.005 1 809 581 T N N 15 114.488 1 810 582 N CA C 13 55.671 1 811582 N CB C 13 37.704 1 812 582 N CO C 13 177.026 1 813 582 N H H 1 8.5591 814 582 N N N 15 124.721 1 815 583 G CA C 13 45.005 1 816 583 G CO C13 174.64 1 817 583 G H H 1 8.964 1 818 583 G N N 15 113.066 1 819 584 WCA C 13 58.735 1 820 584 W CB C 13 27.343 1 821 584 W CO C 13 177.331 1822 584 W H H 1 7.891 1 823 584 W HE1 H 1 10.207 1 824 584 W N N 15120.551 1 825 584 W NE1 N 15 130.072 1 826 585 Q CA C 13 54.336 1 827585 Q CB C 13 32.617 1 828 585 Q CO C 13 173.396 1 829 585 Q H H 1 8.321 830 585 Q N N 15 120.47 1 831 586 L CA C 13 52.787 1 832 586 L CB C 1341.572 1 833 586 L CD1 C 13 24.58 1 834 586 L CD2 C 13 24.177 1 835 586L CO C 13 176.073 1 836 586 L H H 1 8.257 1 837 586 L HD1 H 1 0.875 1838 586 L HD2 H 1 1.06 1 839 586 L N N 15 122.471 1 840 587 F CA C 1356.274 1 841 587 F CB C 13 41.98 1 842 587 F CO C 13 174.71 1 843 587 FH H 1 9.007 1 844 587 F N N 15 119.628 1 845 588 S CA C 13 57.584 1 846588 S CB C 13 64.741 1 847 588 S CO C 13 174.522 1 848 588 S H H 1 8.551 849 588 S N N 15 115.508 1 850 589 K CA C 13 54.504 1 851 589 K CB C13 30.677 1 852 589 K CO C 13 176.928 1 853 589 K H H 1 8.375 1 854 589K N N 15 123.879 1 855 590 K CA C 13 55.59 1 856 590 K CB C 13 32.654 1857 590 K CO C 13 178.646 1 858 590 K H H 1 9.16 1 859 590 K N N 15124.442 1 860 591 S CA C 13 60.73 1 861 591 S CB C 13 62.469 1 862 591 SCO C 13 175.07 1 863 591 S H H 1 8.771 1 864 591 S N N 15 116.878 1 865592 Q CA C 13 56.481 1 866 592 Q CB C 13 27.319 1 867 592 Q CO C 13177.119 1 868 592 Q H H 1 7.787 1 869 592 Q N N 15 114.421 1 870 593 ECA C 13 56.788 1 871 593 E CB C 13 31.514 1 872 593 E CO C 13 176.185 1873 593 E H H 1 8.147 1 874 593 E N N 15 116.571 1 875 594 I CA C 1357.233 1 876 594 I CB C 13 39.464 1 877 594 I H H 1 7.099 1 878 594 I NN 15 111.638 1 879 596 Q CA C 13 52.641 1 880 596 Q CB C 13 31.367 1 881596 Q CO C 13 176.998 1 882 596 Q H H 1 8.568 1 883 596 Q N N 15 119.7761 884 597 Q CA C 13 53.866 1 885 597 Q CB C 13 28.195 1 886 597 Q CO C13 175.677 1 887 597 Q H H 1 8.67 1 888 597 Q N N 15 118.265 1 889 598 MCA C 13 55.496 1 890 598 M CB C 13 33.978 1 891 598 M CO C 13 175.887 1892 598 M H H 1 9.45 1 893 598 M N N 15 118.496 1 894 599 N CA C 1351.768 1 895 599 N CB C 13 39.114 1 896 599 N H H 1 7.565 1 897 599 N NN 15 117.184 1 898 600 G H H 1 9.054 1 899 600 G N N 15 114.081 1 900601 S CA C 13 58.424 1 901 601 S CB C 13 60.647 1 902 601 S H H 1 7.8551 903 601 S N N 15 114.866 1 904 602 D CA C 13 55.181 1 905 602 D CB C13 40.81 1 906 602 D CO C 13 178.539 1 907 602 D H H 1 7.257 1 908 602 DN N 15 118.282 1 909 603 C CA C 13 60.678 1 910 603 C CB C 13 28.27 1911 603 C CO C 13 175.783 1 912 603 C H H 1 7.715 1 913 603 C N N 15121.968 1 914 604 G CA C 13 46.862 1 915 604 G CO C 13 175.076 1 916 604G H H 1 8.736 1 917 604 G N N 15 109.643 1 918 605 M CA C 13 54.421 1919 605 M CB C 13 28.885 1 920 605 M CO C 13 178.831 1 921 605 M H H 16.973 1 922 605 M N N 15 118.381 1 923 606 F CA C 13 63.186 1 924 606 FCB C 13 37.346 1 925 606 F CO C 13 175.928 1 926 606 F H H 1 8.265 1 927606 F N N 15 118.962 1 928 607 A CA C 13 55.826 1 929 607 A CB C 1315.841 1 930 607 A CO C 13 179.705 1 931 607 A H H 1 7.745 1 932 607 A NN 15 118.262 1 933 608 C CA C 13 64.555 1 934 608 C CB C 13 26.489 1 935608 C CO C 13 176.344 1 936 608 C H H 1 7.15 1 937 608 C N N 15 111.3271 938 609 K CA C 13 55.983 1 939 609 K CB C 13 28.003 1 940 609 K CO C13 180.898 1 941 609 K H H 1 8.114 1 942 609 K N N 15 117.546 1 943 610Y CA C 13 58.081 1 944 610 Y CB C 13 36.268 1 945 610 Y CO C 13 177.9421 946 610 Y H H 1 9.584 1 947 610 Y N N 15 121.342 1 948 611 A CA C 1355.284 1 949 611 A CB C 13 17.36 1 950 611 A CO C 13 179.296 1 951 611 AH H 1 7.3 1 952 611 A N N 15 118.235 1 953 612 D CA C 13 57.496 1 954612 D CB C 13 40.809 1 955 612 D CO C 13 177.083 1 956 612 D H H 1 8.2851 957 612 D N N 15 119.051 1 958 613 C CA C 13 64.249 1 959 613 C CB C13 26.401 1 960 613 C CO C 13 177.033 1 961 613 C H H 1 7.283 1 962 613C N N 15 114.15 1 963 614 I CA C 13 64.188 1 964 614 I CB C 13 38.287 1965 614 I CO C 13 180.531 1 966 614 I H H 1 8.523 1 967 614 I N N 15119.103 1 968 615 T CB C 13 67.658 1 969 615 T CO C 13 174.525 1 970 615T H H 1 8.251 1 971 615 T N N 15 108.325 1 972 616 K CA C 13 55.419 1973 616 K CB C 13 31.971 1 974 616 K CO C 13 175.509 1 975 616 K H H 17.204 1 976 616 K N N 15 118.784 1 977 617 D CA C 13 55.028 1 978 617 DCB C 13 38.871 1 979 617 D CO C 13 174.882 1 980 617 D H H 1 7.982 1 981617 D N N 15 117.696 1 982 618 R CA C 13 51.906 1 983 618 R CB C 1330.614 1 984 618 R CO C 13 173.638 1 985 618 R H H 1 7.893 1 986 618 R NN 15 116.707 1 987 620 I CA C 13 62.112 1 988 620 I CB C 13 35.731 1 989620 I CO C 13 177.018 1 990 620 I H H 1 8.465 1 991 620 I N N 15 121.9151 992 621 N CA C 13 52.404 1 993 621 N CB C 13 38.108 1 994 621 N H H 17.952 1 995 621 N N N 15 126.058 1 996 622 F CA C 13 54.399 1 997 622 FCB C 13 41.066 1 998 622 F CO C 13 173.442 1 999 622 F H H 1 6.573 11000 622 F N N 15 114.01 1 1001 623 T CA C 13 59.922 1 1002 623 T CB C13 73.255 1 1003 623 T H H 1 11.019 1 1004 623 T N N 15 112.666 1 1005624 Q CA C 13 57.991 1 1006 624 Q CB C 13 28.115 1 1007 624 Q CO C 13177.817 1 1008 624 Q H H 1 9.841 1 1009 624 Q N N 15 118.693 1 1010 625Q CA C 13 57.772 1 1011 625 Q CB C 13 27.232 1 1012 625 Q CO C 13177.118 1 1013 625 Q H H 1 8.35 1 1014 625 Q N N 15 118.528 1 1015 626 HCA C 13 59.023 1 1016 626 H CB C 13 32.018 1 1017 626 H CO C 13 175.1951 1018 626 H H H 1 7.69 1 1019 626 H N N 15 116.12 1 1020 627 M CA C 1358.703 1 1021 627 M CB C 13 29.282 1 1022 627 M CO C 13 175.481 1 1023627 M H H 1 7.581 1 1024 627 M N N 15 117.619 1 1025 629 Y CA C 1359.898 1 1026 629 Y CB C 13 36.935 1 1027 629 Y CO C 13 176.303 1 1028629 Y H H 1 7.455 1 1029 629 Y N N 15 119.231 1 1030 630 F CA C 1357.548 1 1031 630 F CO C 13 179.707 1 1032 630 F H H 1 8.72 1 1033 630 FN N 15 118.661 1 1034 631 R CA C 13 59.879 1 1035 631 R CB C 13 29.774 11036 631 R CO C 13 177.119 1 1037 631 R H H 1 8.743 1 1038 631 R N N 15121.276 1 1039 632 K CB C 13 32.019 1 1040 632 K CO C 13 178.005 1 1041632 K H H 1 7.027 1 1042 632 K N N 15 115.472 1 1043 633 R CA C 13 59.111 1044 633 R CB C 13 30.377 1 1045 633 R H H 1 8.483 1 1046 633 R N N 15116.56 1 1047 634 M CA C 13 57.95 1 1048 634 M CB C 13 31.937 1 1049 634M H H 1 8.212 1 1050 634 M N N 15 116.933 1 1051 635 V CA C 13 66.729 11052 635 V CB C 13 30.848 1 1053 635 V CG1 C 13 22.182 1 1054 635 V CG2C 13 24.307 1 1055 635 V CO C 13 176.781 1 1056 635 V H H 1 7.401 1 1057635 V HG1 H 1 0.511 1 1058 635 V HG2 H 1 1.078 1 1059 635 V N N 15117.807 1 1060 636 W CA C 13 63.021 1 1061 636 W CB C 13 28.855 1 1062636 W CO C 13 177.991 1 1063 636 W H H 1 6.945 1 1064 636 W HE1 H 110.208 1 1065 636 W N N 15 117.761 1 1066 636 W NE1 N 15 131.254 1 1067637 E CA C 13 59.634 1 1068 637 E CB C 13 29.54 1 1069 637 E CO C 13179.549 1 1070 637 E H H 1 8.942 1 1071 637 E N N 15 118.312 1 1072 638I CA C 13 65.061 1 1073 638 I CB C 13 36.564 1 1074 638 I CO C 13178.793 1 1075 638 I H H 1 8.516 1 1076 638 I N N 15 118.151 1 1077 639L CA C 13 57.489 1 1078 639 L CB C 13 40.751 1 1079 639 L CD1 C 1325.065 1 1080 639 L CD2 C 13 22.821 1 1081 639 L H H 1 8.021 1 1082 639L HD1 H 1 0.577 1 1083 639 L HD2 H 1 0.498 1 1084 639 L N N 15 119.857 11085 640 H CA C 13 55.633 1 1086 640 H CB C 13 26.845 1 1087 640 H H H 17.758 1 1088 640 H N N 15 112.218 1 1089 641 R CA C 13 56.955 1 1090 641R CB C 13 25.944 1 1091 641 R CO C 13 174.773 1 1092 641 R H H 1 7.879 11093 641 R N N 15 122.564 1 1094 642 K CA C 13 54.648 1 1095 642 K CB C13 34.915 1 1096 642 K CO C 13 172.886 1 1097 642 K H H 1 8.22 1 1098642 K N N 15 121.148 1 1099 643 L CA C 13 53.678 1 1100 643 L CB C 1341.237 1 1101 643 L CD1 C 13 27.306 1 1102 643 L CD2 C 13 24.45 1 1103643 L CO C 13 177.594 1 1104 643 L H H 1 8.136 1 1105 643 L HD1 H 10.395 1 1106 643 L HD2 H 1 0.572 1 1107 643 L N N 15 122.205 1 1108 644L CA C 13 55.222 1 1109 644 L CB C 13 41.615 1 1110 644 L CO C 13182.082 1 1111 644 L H H 1 8.863 1 1112 644 L N N 15 130.114 1*referenced using DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) asthe H-1 standard with IUPAC-IUB recommended chemical shift referencingratios. See, Wishart, et al., “1H, 13C and 15N Chemical ShiftReferencing in Biomolecular NMR,” J. Biomol. NMR 6: 135-140 (1995); andMarkley et al., “Recommendations for the Presentation of NMR Structuresof Proteins and Nucleic Acids,”. Pure & Appl. Chem. 70: 117-142 (1998).

TABLE 4 SENP1 C603S-SUMO₁₋₉₂ NMR Chemical Shift Values. Chemical ShiftAmbiguity Index Value Definitions The values other than 1 are used forthose atoms with different chemical shifts that cannot be assigned tostereospecific atoms or to specific residues or chains. Index ValueDefinition 1 Unique (including isolated methyl protons germinal atoms,and geminal methyl groups with identical chemical shifts (e.g. ILE HD11,HD12, HD13 protons) 2 Ambiguity of geminal atoms or geminal methylproton groups (e.g. ASP HB2 and HB3 protons, LEU CD1 and CD2 carbons, orLEU HD11, HD12, HD13 and HD21, HD22, HD23 methyl protons) 3 Aromaticatoms on opposite sides of symmetrical rings (e.g. TYR HE1 and HE2protons) 4 Intraresidue ambiguities (e.g. LYS HG and HD protons or TRPHZ2 and HZ3 protons) 5 Interresidue ambiguities (LYS 12 vs. LYS 27) 6Intermolecular ambiguities (e.g. ASP 31 CA in monomer 1 and ASP 31 CA inmonomer 2 of an asymmetrical homodimer, duplex DNA assignments, or otherassignments that may apply to atoms in one or more molecule in themolecular assembly) 9 Ambiguous, specific ambiguity not defined ChemicalAtom Residue Amino Atom Atom Iso- shift Unique- number number acidcontext type type (ppm)* ness 1 419 E H H 1 7.974 1 2 419 E N N 15121.157 1 3 420 F H H 1 7.947 1 4 420 F N N 15 119.83 1 5 422 E H H 18.562 1 6 422 E N N 15 125.045 1 7 423 I H H 1 8.443 1 8 423 I N N 15123.042 1 9 424 T H H 1 7.558 1 10 424 T N N 15 122.22 1 11 425 E H H 18.835 1 12 425 E N N 15 122.015 1 13 426 E H H 1 8.343 1 14 426 E N N 15119.067 1 15 427 M H H 1 7.295 1 16 427 M N N 15 120.177 1 17 428 E H H1 8.525 1 18 428 E N N 15 119.639 1 19 429 K H H 1 7.793 1 20 429 K N N15 119.161 1 21 430 E H H 1 7.247 1 22 430 E N N 15 120.017 1 23 432 K HH 1 8.269 1 24 432 K N N 15 117.252 1 25 433 D H H 1 7.542 1 26 433 D NN 15 116.02 1 27 434 V CG1 C 13 22.744 1 28 434 V H H 1 7.456 1 29 434 VN N 15 115.391 1 32 434 V HG1 H 1 0.768 1 33 435 F H H 1 7.229 1 34 435F N N 15 118.53 1 35 436 R H H 1 7.082 1 36 436 R N N 15 119.966 1 37437 D H H 1 8.266 1 38 437 D N N 15 120.742 1 39 438 G H H 1 7.994 1 40438 G N N 15 110.561 1 41 439 D H H 1 8.68 1 42 439 D N N 15 121.505 143 440 Q H H 1 8.954 1 44 440 Q N N 15 126.9 1 45 441 D H H 1 7.858 1 46441 D N N 15 115.853 1 47 442 E H H 1 7.08 1 48 442 E N N 15 122.689 149 443 V CG1 C 13 21.595 1 50 443 V CG2 C 13 22.122 1 51 443 V H H 18.563 1 52 443 V N N 15 128.078 1 55 443 V HG1 H 1 0.731 1 58 443 V HG2H 1 0.896 1 59 444 L CD1 C 13 27.192 1 60 444 L H H 1 8.928 1 61 444 L NN 15 128.342 1 64 444 L HD1 H 1 0.609 1 65 445 S H H 1 7.327 1 66 445 SN N 15 112.548 1 67 446 E H H 1 7.889 1 68 446 E N N 15 125.959 1 69 447A H H 1 8.267 1 70 447 A N N 15 125.511 1 71 448 F H H 1 8.549 1 72 448F N N 15 117.045 1 73 449 R H H 1 8.43 1 74 449 R N N 15 112.963 1 75450 L CD1 C 13 26.152 1 76 450 L CD2 C 13 23.171 1 77 450 L H H 1 8.2881 78 450 L N N 15 121.64 1 81 450 L HD1 H 1 0.918 1 84 450 L HD2 H 11.032 1 85 451 T H H 1 8.297 1 86 451 T N N 15 113.505 1 87 452 I H H 18.385 1 88 452 I N N 15 124.62 1 89 453 T H H 1 9.666 1 90 453 T N N 15120.795 1 91 454 R H H 1 8.168 1 92 454 R N N 15 123.055 1 93 455 K H H1 8.41 1 94 455 K N N 15 120.473 1 95 456 D H H 1 7.227 1 96 456 D N N15 118.114 1 97 457 I H H 1 8.102 1 98 457 I N N 15 122.652 1 99 458 Q HH 1 7.821 1 100 458 Q N N 15 119.174 1 101 459 T H H 1 7.826 1 102 459 TN N 15 114.259 1 103 460 L CD1 C 13 26.138 1 104 460 L CD2 C 13 26.003 1105 460 L H H 1 7.18 1 106 460 L N N 15 116.293 1 109 460 L HD1 H 10.845 1 112 460 L HD2 H 1 0.954 1 113 461 D H H 1 7.356 1 114 461 D N N15 121.211 1 115 462 H H H 1 7.659 1 116 462 H N N 15 120.742 1 117 465W H H 1 8.258 1 118 465 W N N 15 121.504 1 119 465 W HE1 H 1 9.997 1 120465 W NE1 H 1 130.62 1 121 466 L CD1 C 13 25.625 1 122 466 L CD2 C 1323.625 1 123 466 L H H 1 7.529 1 124 466 L N N 15 126.826 1 127 466 LHD1 H 1 0.684 1 130 466 L HD2 H 1 0.704 1 131 467 D H H 1 6.937 1 132467 D N N 15 117.87 1 133 468 D H H 1 8.19 1 134 468 D N N 15 115.279 1135 470 I H H 1 7.581 1 136 470 I N N 15 118.739 1 137 471 I H H 1 6.8231 138 471 I N N 15 118.333 1 139 472 D H H 1 8.781 1 140 472 D N N 15116.126 1 141 473 F H H 1 8.248 1 142 473 F N N 15 124.484 1 143 475 M HH 1 8.642 1 144 475 M N N 15 116.267 1 145 476 D H H 1 7.328 1 146 476 DN N 15 118.167 1 147 477 M H H 1 7.588 1 148 477 M N N 15 122.218 1 149478 L CD1 C 13 27.574 1 150 478 L CD2 C 13 22.365 1 151 478 L H H 17.689 1 152 478 L N N 15 121.041 1 155 478 L HD1 H 1 0.707 1 158 478 LHD2 H 1 0.458 1 159 479 M H H 1 7.581 1 160 479 M N N 15 120.182 1 161480 E H H 1 8.04 1 162 480 E N N 15 123.808 1 163 481 R H H 1 7.872 1164 481 R N N 15 121.587 1 165 482 S H H 1 7.133 1 166 482 S N N 15114.199 1 167 483 K H H 1 6.896 1 168 483 K N N 15 119.755 1 169 484 E HH 1 8.035 1 170 484 E N N 15 122.017 1 171 485 K H H 1 8.183 1 172 485 KN N 15 122.621 1 173 486 G H H 1 8.674 1 174 486 G N N 15 112.424 1 175487 L CD1 C 13 25.976 1 176 487 L CD2 C 13 23.426 1 177 487 L H H 17.277 1 178 487 L N N 15 122.648 1 181 487 L HD1 H 1 0.802 1 184 487 LHD2 H 1 0.853 1 185 489 S H H 1 9.049 1 186 489 S N N 15 119.012 1 187490 V CG1 C 13 21.236 1 188 490 V CG2 C 13 23.244 1 189 490 V H H 17.325 1 190 490 V N N 15 119.713 1 191 490 V HG1 H 1 0.583 1 196 490 VHG2 H 1 0.912 1 197 491 H H H 1 8.716 1 198 491 H N N 15 125.135 1 199492 A H H 1 7.396 1 200 492 A N N 15 130.645 1 201 494 D H H 1 8.676 1202 494 D N N 15 117.371 1 203 495 T H H 1 8.641 1 204 495 T N N 15112.625 1 205 497 F H H 1 7.868 1 206 497 F N N 15 122.062 1 207 498 F HH 1 9.932 1 208 498 F N N 15 121.373 1 209 499 T H H 1 6.901 1 210 499 TN N 15 113.079 1 211 500 K H H 1 7.689 1 212 500 K N N 15 123.985 1 213501 L CD1 C 13 21.431 1 214 501 L CD2 C 13 26.226 1 217 501 L HD1 H 10.589 1 220 501 L HD2 H 1 0.244 1 221 502 K H H 1 8.034 1 222 502 K N N15 118.063 1 223 503 T H H 1 7.409 1 224 503 T N N 15 109.905 1 225 504A H H 1 8.332 1 226 504 A N N 15 125.057 1 227 505 G H H 1 7.268 1 228505 G N N 15 109.015 1 229 506 Y H H 1 8.399 1 230 506 Y N N 15 118.7991 231 507 Q H H 1 8.555 1 232 507 Q N N 15 114.738 1 233 508 A H H 17.049 1 234 508 A N N 15 118.763 1 235 509 V CG1 C 13 19.211 1 236 509 VCG2 C 13 20.045 1 237 509 V H H 1 6.759 1 238 509 V N N 15 105.237 1 241509 V HG1 H 1 0.198 1 244 509 V HG2 H 1 0.47 1 245 510 K H H 1 7.119 1246 510 K N N 15 128.018 1 247 511 R H H 1 8.684 1 248 511 R N N 15117.231 1 249 512 W H H 1 8.542 1 250 512 W N N 15 120.625 1 251 512 WHE1 H 1 9.942 1 252 512 W NE1 H 1 130.341 1 253 513 T H H 1 7.069 1 254513 T N N 15 106.003 1 255 514 K H H 1 7.238 1 256 514 K N N 15 121.3581 257 515 K H H 1 8.487 1 258 515 K N N 15 116.476 1 259 516 V CG1 C 1322.32 1 260 516 V CG2 C 13 19.555 1 261 516 V H H 1 7.299 1 262 516 V NN 15 119.473 1 265 516 V HG1 H 1 1.018 1 268 516 V HG2 H 1 0.836 1 269517 D H H 1 8.389 1 270 517 D N N 15 124.991 1 271 518 V CG1 C 13 21.9721 272 518 V CG2 C 13 17.937 1 273 518 V H H 1 8.864 1 274 518 V N N 15122.483 1 277 518 V HG1 H 1 0.73 1 280 518 V HG2 H 1 0.268 1 281 519 F HH 1 7.156 1 282 519 F N N 15 111.705 1 283 520 S H H 1 7.418 1 284 520 SN N 15 114.604 1 285 521 V CG1 C 13 21.606 1 286 521 V CG2 C 13 19.292 1287 521 V H H 1 6.57 1 288 521 V N N 15 114.783 1 291 521 V HG1 H 10.698 1 294 521 V HG2 H 1 0.757 1 295 522 D H H 1 8.102 1 296 522 D N N15 121.096 1 297 523 I H H 1 8.117 1 298 523 I N N 15 118.339 1 299 524L CD1 C 13 24.558 2 300 524 L CD2 C 13 24.558 2 301 524 L H H 1 9.253 1302 524 L N N 15 122.892 1 305 524 L HG1 H 1 0.841 2 308 524 L HG2 H 10.841 2 309 525 L CD1 C 13 27.708 2 310 525 L CD2 C 13 23.849 1 311 525L H H 1 8.636 1 312 525 L N N 15 121.574 1 315 525 L HD1 H 1 0.775 1 318525 L HD2 H 1 0.735 1 319 526 V CG1 C 13 20.514 1 320 526 V CG2 C 1319.367 1 323 526 V HG1 H 1 −0.557 1 326 526 V HG2 H 1 0.332 1 327 528 IH H 1 8.599 1 328 528 I N N 15 125.838 1 329 529 H H H 1 9.049 1 330 529H N N 15 130.138 1 331 530 L CD1 C 13 25.956 1 332 530 L CD2 C 13 23.9451 333 530 L H H 1 8.536 1 334 530 L N N 15 131.927 1 337 530 L HD1 H 10.891 1 340 530 L HD2 H 1 0.759 1 341 531 G H H 1 8 1 342 531 G N N 15116.007 1 343 532 V CG1 C 13 20.988 1 344 532 V CG2 C 13 17.895 1 347532 V HG1 H 1 0.507 1 350 532 V HG2 H 1 0.396 1 351 533 H H H 1 7.652 1352 533 H N N 15 123.973 1 353 534 W H H 1 7.787 1 354 534 W N N 15125.281 1 355 534 W HE1 H 1 9.36 1 356 534 W NE1 H 1 128.566 1 357 535 CH H 1 9.374 1 358 535 C N N 15 118.493 1 359 536 L CD1 C 13 22.488 1 360536 L CD2 C 13 26.383 1 363 536 L HD1 H 1 0.708 1 366 536 L HD2 H 10.622 1 367 537 A H H 1 8.735 1 368 537 A N N 15 127.969 1 369 538 V CG1C 13 21.782 1 370 538 V CG2 C 13 22.071 1 371 538 V H H 1 8.971 1 372538 V N N 15 120.496 1 375 538 V HG1 H 1 0.885 1 378 538 V HG2 H 1 0.8311 379 539 V CG1 C 13 20.583 1 380 539 V CG2 C 13 19.613 1 381 539 V H H1 9.299 1 382 539 V N N 15 131.537 1 385 539 V HG1 H 1 0.452 1 388 539 VHG2 H 1 0.894 1 389 540 D H H 1 8.849 1 390 540 D N N 15 127.571 1 391541 F H H 1 9.394 1 392 541 F N N 15 125.014 1 393 542 R H H 1 8.636 1394 542 R N N 15 118.378 1 395 543 K H H 1 6.667 1 396 543 K N N 15115.058 1 397 544 K H H 1 7.337 1 398 544 K N N 15 116.66 1 399 545 D HH 1 7.143 1 400 545 D N N 15 114.477 1 401 546 I H H 1 8.348 1 402 546 IN N 15 121.213 1 403 547 T H H 1 8.68 1 404 547 T N N 15 122.503 1 405548 Y H H 1 8.599 1 406 548 Y N N 15 129.81 1 407 549 Y H H 1 8.988 1408 549 Y N N 15 126.841 1 409 550 D H H 1 7.986 1 410 550 D N N 15124.111 1 411 551 S H H 1 9.257 1 412 551 S N N 15 123.506 1 413 552 M HH 1 9.118 1 414 552 M N N 15 122.883 1 415 553 G H H 1 7.765 1 416 553 GN N 15 110.571 1 417 554 G H H 1 7.593 1 418 554 G N N 15 108.922 1 419555 I H H 1 7.986 1 420 555 I N N 15 119.104 1 421 556 D H H 1 7.676 1422 556 D N N 15 125.208 1 423 557 D H H 1 8.221 1 424 557 D N N 15122.087 1 425 558 E H H 1 8.444 1 426 558 E N N 15 121.771 1 427 559 A HH 1 7.431 1 428 559 A N N 15 121.623 1 429 560 C H H 1 6.749 1 430 560 CN N 15 112.908 1 431 561 R H H 1 7.935 1 432 561 R N N 15 121.153 1 433562 I H H 1 8.494 1 434 562 I N N 15 121.654 1 435 563 L CD1 C 13 26.2511 436 563 L H H 1 7.51 1 437 563 L N N 15 121.675 1 440 563 L HD1 H 10.705 1 441 564 L CD1 C 13 23.21 1 442 564 L CD2 C 13 25.851 1 443 564 LH H 1 7.927 1 444 564 L N N 15 123.395 1 447 564 L HD1 H 1 0.527 1 450564 L HD2 H 1 0.857 1 451 565 Q H H 1 7.814 1 452 565 Q N N 15 117.094 1453 566 Y H H 1 8.072 1 454 566 Y N N 15 122.424 1 455 567 L CD1 C 1326.248 1 456 567 L H H 1 7.659 1 457 567 L N N 15 119.901 1 460 567 LHD1 H 1 0.295 1 461 568 K H H 1 7.303 1 462 568 K N N 15 117.193 1 463569 Q H H 1 7.605 1 464 569 Q N N 15 120.146 1 465 570 E H H 1 8.815 1466 570 E N N 15 125.031 1 467 571 S H H 1 7.999 1 468 571 S N N 15113.963 1 469 572 I H H 1 6.963 1 470 572 I N N 15 120.839 1 471 573 D HH 1 8.221 1 472 573 D N N 15 120.607 1 473 574 K H H 1 8.471 1 474 574 KN N 15 110.577 1 475 575 K H H 1 7.695 1 476 575 K N N 15 115.597 1 477580 D H H 1 7.94 1 478 580 D N N 15 129.176 1 479 581 T H H 1 7.92 1 480581 T N N 15 115.295 1 481 582 D H H 1 8.477 1 482 582 D N N 15 125.7591 483 584 W HE1 H 1 10.14 1 484 584 W NE1 H 1 131.131 1 485 585 Q H H 18.228 1 486 585 Q N N 15 121.5 1 487 586 L CD1 C 13 24.774 1 488 586 L HH 1 8.179 1 489 586 L N N 15 123.493 1 492 586 L HD1 H 1 0.902 1 493 587F H H 1 8.927 1 494 587 F N N 15 120.564 1 495 588 S H H 1 8.491 1 496588 S N N 15 116.853 1 497 589 K H H 1 8.272 1 498 589 K N N 15 125.1481 499 590 K H H 1 9.035 1 500 590 K N N 15 125.714 1 501 591 S H H 18.628 1 502 591 S N N 15 117.949 1 503 592 Q H H 1 7.72 1 504 592 Q N N15 115.49 1 505 593 E H H 1 8.054 1 506 593 E N N 15 117.488 1 507 594 IH H 1 7.053 1 508 594 I N N 15 112.806 1 509 596 Q H H 1 8.456 1 510 596Q N N 15 120.73 1 511 597 Q H H 1 8.576 1 512 597 Q N N 15 119.759 1 513598 M H H 1 9.401 1 514 598 M N N 15 120.151 1 515 599 D H H 1 7.357 1516 599 D N N 15 116.972 1 517 602 D H H 1 7.383 1 518 602 D N N 15119.804 1 519 603 S H H 1 8.061 1 520 603 S N N 15 121.228 1 521 604 G HH 1 8.814 1 522 604 G N N 15 109.019 1 523 605 M H H 1 6.87 1 524 605 MN N 15 119.181 1 525 606 F H H 1 8.087 1 526 606 F N N 15 120.065 1 527607 A H H 1 7.948 1 528 607 A N N 15 119.608 1 529 608 C H H 1 7.095 1530 608 C N N 15 112.346 1 531 609 K H H 1 7.948 1 532 609 K N N 15118.448 1 533 610 Y H H 1 9.542 1 534 610 Y N N 15 122.405 1 535 611 A HH 1 7.333 1 536 611 A N N 15 119.249 1 537 612 D H H 1 8.263 1 538 612 DN N 15 120.131 1 539 613 C H H 1 7.203 1 540 613 C N N 15 115.162 1 541614 I H H 1 8.484 1 542 614 I N N 15 120.137 1 543 615 T H H 1 8.165 1544 615 T N N 15 109.158 1 545 616 K H H 1 7.126 1 546 616 K N N 15119.661 1 547 617 D H H 1 7.896 1 548 617 D N N 15 118.551 1 549 618 R HH 1 7.855 1 550 618 R N N 15 117.684 1 551 620 I H H 1 8.365 1 552 620 IN N 15 122.909 1 553 621 D H H 1 7.872 1 554 621 D N N 15 127.17 1 555622 F H H 1 6.479 1 556 622 F N N 15 114.836 1 557 623 T H H 1 10.893 1558 623 T N N 15 113.615 1 559 624 Q H H 1 9.761 1 560 624 Q N N 15119.808 1 561 625 Q H H 1 8.292 1 562 625 Q N N 15 119.007 1 563 626 H HH 1 7.627 1 564 626 H N N 15 117.04 1 565 627 M H H 1 7.554 1 566 627 MN N 15 118.727 1 567 629 Y H H 1 7.389 1 568 629 Y N N 15 120.089 1 569630 F H H 1 8.629 1 570 630 F N N 15 119.732 1 571 631 R H H 1 8.753 1572 631 R N N 15 122.645 1 573 632 K H H 1 6.955 1 574 632 K N N 15116.472 1 575 633 R H H 1 8.412 1 576 633 R N N 15 117.619 1 577 634 M HH 1 8.154 1 578 634 M N N 15 117.932 1 579 635 V CG1 C 13 22.402 1 580635 V CG2 C 13 24.472 1 581 635 V H H 1 7.322 1 582 635 V N N 15 118.681 585 635 V HG1 H 1 0.533 1 588 635 V HG2 H 1 1.092 1 589 636 W H H 16.859 1 590 636 W N N 15 118.812 1 591 636 W HE1 H 1 10.124 1 592 636 WNE1 H 1 132.344 1 593 637 E H H 1 8.839 1 594 637 E N N 15 119.355 1 595638 I H H 1 8.41 1 596 638 I N N 15 119.139 1 597 639 L CD1 C 13 25.3131 598 639 L CD2 C 13 22.916 1 599 639 L H H 1 7.977 1 600 639 L N N 15120.642 1 603 639 L HD1 H 1 0.596 1 606 639 L HD2 H 1 0.514 1 607 640 HH H 1 7.669 1 608 640 H N N 15 113.238 1 609 641 R H H 1 7.794 1 610 641R N N 15 123.547 1 611 642 K H H 1 8.152 1 612 642 K N N 15 122.071 1613 643 L CD1 C 13 27.478 1 614 643 L CD2 C 13 24.632 1 615 643 L H H 18.058 1 616 643 L N N 15 123.331 1 619 643 L HD1 H 1 0.412 1 622 643 LHD2 H 1 0.573 1 623 644 L H H 1 8.748 1 624 644 L N N 15 131.146 1*referenced using DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) asthe H-1 standard with IUPAC-IUB recommended chemical shift referencingratios. See, Wishart, et al., “1H, 13C and 15N Chemical ShiftReferencing in Biomolecular NMR,” J. Biomol. NMR 6: 135-140 (1995); andMarkley et al., “Recommendations for the Presentation of NMR Structuresof Proteins and Nucleic Acids,”. Pure & Appl. Chem. 70: 117-142 (1998).

Sequence Listing SEQ ID NO: 1 Isoform 1 SENP1 MDDIADRMRM DAGEVTLVNH NSVFKTHLLP QTGFPEDQLS LSDQQILSSR QGHLDRSFTC STRSAAYNPS YYSDNPSSDS FLGSGDLRTF GQSANGQWRN STPSSSSSLQ KSRNSRSLYL ETRKTSSGLS NSFAGKSNHH CHVSAYEKSF PIKPVPSPSW SGSCRRSLLS PKKTQRRHVS TAEETVQEEE REIYRQLLQM VTGKQFTIAK PTTHFPLHLS RCLSSSKNTL KDSLFKNGNS CASQIIGSDT SSSGSASILT NQEQLSHSVY SLSSYTPDVA FGSKDSGTLH HPHHHHSVPH QPDNLAASNT QSEGSDSVIL LKVKDSQTPT PSSTFFQAEL WIKELTSVYD SRARERLRQI EEQKALALQL QNQRLQEREH SVHDSVELHL RVPLEKEIPV TVVQETQKKG HKLTDSEDEF PEITEEMEKE IKNVFRNGNQ DEVLSEAFRL TITRKDIQTL NHLNWLNDEI INFYMNMLME RSKEKGLPSV HAFNTFFFTK LKTAGYQAVK RWTKKVDVFS VDILLVPIHL GVHWCLAVVD FRKKNITYYD SMGGINNEAC RILLQYLKQE SIDKKRKEFD TNGWQLFSKK SQEIPQQMNG SDCGMFACKY ADCITKDRPI NFTQQHMPYF RKRMVWEILH  RKLL SEQ ID NO: 2 Isoform 2 SENP1 MDDIADRMRM DAGEVTLVNH NSVFKTHLLP QTGFPEDQLS LSDQQILSSR QGHLDRSFTC STRSAAYNPS YYSDNPSSDS FLGSGDLRTF GQSANGQWRN STPSSSSSLQ KSRNSRSLYL ETRKTSSGLS NSFAGKSNHH CHVSAYEKSF PIKPVPSPSW SGSCRRSLLS PKKTQRRHVS TAEETVQEEE REIYRQLLQM VTGKQFTIAK PTTHFPLHLS RCLSSSKNTL KDSLFKNGNS CASQIIGSDT SSSGSASILT NQEQLSHSVY SLSSYTPDVA FGSKDSGTLH HPHHHHSVPH QPDNLAASNT QSEGSDSVIL LKVKDSQTPT PSSTFFQAEL WIKELTSVYD SRARERLRQI EEQKALALQL QNQRLQEREH SVHDSVELHL RVPLEKEIPV TVVQETQKKG HKLTDSEDEF PEITEEMEKE IKNVFRNGNQ DEVLSEAFRL TITRKDIQTL NHLNWLNDEI INFYMNMLME RSKEKGLPSV HAFNTFFFTK LKTAGYQAVK RWTKKVDVFS VDILLVPIHL GVHWCLAVVD FRKKNITYYD SMGGINNEAC RILLQYLKQE SIDKKRKEFD TNGWQLFSKK SQIPQQMNGS DCGMFACKYA DCITKDRPIN FTQQHMPYFR KRMVWEILHR  KLL SEQ ID NO: 3 (Isoform 1) C-Terminal Region SENP1 EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLNDEI INFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDVFSVDI LLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLKQESIDKKRKE FDTNGWQLFSKKSQEIPQQMNGSDCGMFACKYADCITKDRPINFTQQHMPYFRK  RMVWEILHRKLL SEQ ID NO: 4 (Isoform 1) C-Terminal Region SENP1 C6035 EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLNDEI INFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDVFSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLKQESIDKKRKE FDTNGWQLFSKKSQEIPQQMNGSDSGMFACKYADCITKDRPINFTQQHMPYFRK  RMVWEILHRKLL SEQ ID NO: 5 (Isoform 2) C-Terminal Region SENP1 EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLNDEI INFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDVFSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLKQESIDKKRKE FDTNGWQLFSKKSQIPQQMNGSDCGMFACKYADCITKDRPINFTQQHMPYFRKR  MVWEILHRKLL SEQ ID NO: 6 (Isoform 1) Protease Region 450-613 SENP1 LTITRKDIQTLNHLNWLNDEIINFYMNMLMERSKEKGLPSVHAFNTFFFTK LKTAGYQAVKRWTKKVDVFSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMG GINNEACRILLQYLKQESIDKKRKEFDTNGWQLFSKKSQEIPQQMNGSDCGMFA  CKYADC SEQ ID NO: 7 (Isoform 1) Protease Region 450-613 SENP1 C603S LTITRKDIQTLNHLNWLNDEIINFYMNMLMERSKEKGLPSVHAFNTFFFTK LKTAGYQAVKRWTKKVDVFSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMG GINNEACRILLQYLKQESIDKKRKEFDTNGWQLFSKKSQEIPQQMNGSDSGMFA  CKYADC SEQ ID NO: 8 SUMO1  MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKES YCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV SEQ ID NO: 9 SUMO1 (1-92) MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQR QGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQ 

EMBODIMENTS Embodiment 1

A method of detecting binding of an SENP1 polypeptide to a compound, themethod comprising:

(i) contacting an SENP1 polypeptide with a compound;

(ii) allowing the compound to bind to the SENP1 polypeptide, therebyforming a SENP1-compound complex;

(iii) detecting the SENP1-compound complex using nuclear magneticresonance, thereby detecting binding of the SENP1 polypeptide to thecompound.

Embodiment 2

The method of embodiment 1, wherein the detecting comprises determininga chemical shift for an amino acid in an active site of the SENP1polypeptide.

Embodiment 3

The method of embodiment 2, wherein the chemical shift in the presenceof the compound is changed relative to the corresponding chemical shiftin the absence of the compound.

Embodiment 4

The method of embodiment 2 or 3, wherein the amino acid is an amino acidof SEQ ID NOs:3, 4, 5, 6 or 7.

Embodiment 5

The method of embodiment 2 or 3, wherein the amino acid is selected fromthe group consisting of D550, H533, C603, W465, W534, L466, G531, C535,M552, G554, E469 and Q596.

Embodiment 6

The method of embodiment 2 or 3, wherein the amino acid is S603.

Embodiment 7

The method of embodiment 2 or 3, wherein the amino acid is amino acidresidue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQID NO:1.

Embodiment 8

The method of embodiment 1, wherein the SENP1 polypeptide comprises SEQID NOs:1, 2, 3, 4, 5, 6, or 7.

Embodiment 9

The method of embodiment 1, wherein the SENP1 polypeptide comprisesamino acid residue 603 of SEQ ID NO:1.

Embodiment 10

The method of embodiment 9, wherein the SENP1 polypeptide comprises amutation at amino acid residue 603 of SEQ ID NO:1.

Embodiment 11

The method of embodiment 10, wherein the mutation is C603S.

Embodiment 12

The method of embodiment 1, wherein the SENP1 polypeptide comprisesamino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or596-603 of SEQ ID NO:1.

Embodiment 13

The method of any one of embodiments 1-12, wherein the SENP1 orSENP1-compound complex is bound to a SUMO protein thereby forming aSENP1-SUMO complex or SENP1-SUMO-compound complex.

Embodiment 14

The method of embodiment 13, wherein the SUMO protein is a truncatedSUMO protein.

Embodiment 15

The method of embodiment 2, wherein the active site is a catalyticallyactive site.

Embodiment 16

The method of embodiment 2, wherein the active site is a site that bindsto the SUMO protein.

Embodiment 17

The method of any one of embodiments 1-16, wherein the compound is asmall molecule.

Embodiment 18

The method of any one of embodiments 1 or 8-17, wherein the detectingcomprises producing an NMR spectra of the SENP1-compound complex andidentifying a change in the NMR spectra relative to the absence of thecompound.

Embodiment 19

The method of embodiment 18, wherein the change is a change in thechemical shift of an amino acid of SEQ ID NOs:3, 4, 5, 6 or 7.

Embodiment 20

The method of embodiment 18, wherein the change is a change in thechemical shift of an amino acid selected from the group consisting ofD550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 andQ596.

Embodiment 21

The method of embodiment 18, wherein the change is a change in thechemical shift of the amino acid S603.

Embodiment 22

The method of embodiment 18, wherein the change is a change in thechemical shift of an amino acid residue 440-455, 463-473, 493-515,529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 23

An aqueous composition comprising an SENP1 polypeptide at a pH fromabout 6.0 to about 7.5.

Embodiment 24

The aqueous composition of embodiment 23, wherein the pH is about 6.8.

Embodiment 25

The aqueous composition of embodiment 23 or 24, further comprising abuffering agent, reducing agent, solvent, a base, or combinationsthereof.

Embodiment 26

The aqueous composition of any one of embodiments 23-25, furthercomprising sodium phosphate, dimethyl sulfoxide, D2O, sodium azide,dithiothreitol or combinations thereof.

Embodiment 27

The aqueous composition of embodiment 26, wherein the sodium phosphateis present at about 20 mM.

Embodiment 28

The aqueous composition of any one of embodiments 23-27, wherein theSENP1 polypeptide comprises SEQ ID NO:1, 2, 3, 4, 5, 6, or 7.

Embodiment 29

The aqueous composition of any one of embodiments 23-27, wherein theSENP1 polypeptide comprises amino acid residues 440-455, 463-473,493-515, 529-535, 550-554, or 596-603 numbered relative to SEQ ID NO:1.

Embodiment 30

The aqueous composition of any one of embodiments 23-29, wherein theSENP1 polypeptide is bound to a SUMO protein thereby forming aSENP1-SUMO complex.

Embodiment 31

The aqueous composition of any one of embodiments 23-29, wherein theSENP1 polypeptide is bound to a compound thereby forming aSENP1-compound complex.

Embodiment 32

The aqueous composition of embodiment 31, wherein the SENP1 polypeptideis bound to a SUMO protein thereby forming a SENP1-SUMO-compoundcomplex.

Embodiment 33

The aqueous composition of embodiment 30 or 32, wherein the SUMO proteinis a truncated SUMO protein.

Embodiment 34

An NMR apparatus comprising an NMR sample container for NMR analysis,the NMR sample container comprising the aqueous composition of any oneof embodiments 23-33.

Embodiment 35

A method of screening for an inhibitor of SENP1 comprising contacting acomposition comprising an SENP1 polypeptide with a test compound anddetecting whether the test compound binds the SENP1 polypeptide orfragment thereof by nuclear magnetic resonance.

Embodiment 36

The method of embodiment 35, wherein the detecting comprises determininga chemical shift for an amino acid in an active site of the SENP1polypeptide.

Embodiment 37

The method of embodiment 36, wherein the amino acid is an amino acid ofSEQ ID NOs:3, 4, 5, 6 OR 7.

Embodiment 38

The method of embodiment 36, wherein the amino acid is selected from thegroup consisting of D550, H533, C603, W465, W534, L466, G531, C535,M552, G554, E469 and Q596.

Embodiment 39

The method of embodiment 36, wherein the amino acid is S603.

Embodiment 40

The method of embodiment 36, wherein the amino acid is amino acidresidue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQID NO:1.

Embodiment 41

The method of embodiment 35, wherein the SENP1 polypeptide comprises SEQID NOs:1, 2, 3, 4, 5, 6, or 7.

Embodiment 42

The method of embodiment 35, wherein the SENP1 polypeptide comprisesamino acid residue 603 of SEQ ID NO:1.

Embodiment 43

The method of embodiment 42, wherein the SENP1 polypeptide comprises amutation at amino acid residue 603 of SEQ ID NO:1.

Embodiment 44

The method of embodiment 43, wherein the mutation is C603S.

Embodiment 45

The method of embodiment 35, wherein the SENP1 polypeptide comprisesamino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or596-603 of SEQ ID NO:1.

Embodiment 46

The method of any one of embodiments 35-45, wherein the SENP1polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMOcomplex.

Embodiment 47

The method of embodiment 46, wherein the SUMO protein is a truncatedSUMO protein.

Embodiment 48

The method of any one of embodiments 35-47, wherein the chemical shiftin the presence of the test compound is changed relative to thecorresponding chemical shift in the absence of the test compound.

Embodiment 49

The method of any one of embodiments 35-47, wherein the SENP1 binds thecompound forming an SENP1-compound complex and the detecting comprisesproducing an NMR spectra of the SENP1-compound complex and identifying achange in the NMR spectra relative to the absence of the compound.

Embodiment 50

The method of embodiment 49, wherein the change is a change in thechemical shift of an amino acid of SEQ ID NOs:3, 4, 5, 6 or 7.

Embodiment 51

The method of embodiment 49, wherein the change is a change in thechemical shift of an amino acid selected from the group consisting ofD550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 andQ596.

Embodiment 52

The method of embodiment 49, wherein the change is a change in thechemical shift of the amino acid S603.

Embodiment 53

The method of embodiment 49, wherein the change is a change in thechemical shift of an amino acid residue 440-455, 463-473, 493-515,529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 54

The method of embodiment 49, wherein the change is a change in thechemical shift of an amino acid in the active site of SENP1.

Embodiment 55

The method of embodiment 54, wherein the active site is a catalyticallyactive site.

Embodiment 56

The method of embodiment 54, wherein the active site is a site thatbinds to the SUMO protein.

Embodiment 57

The method of any one of embodiments 35-56, wherein the test compound isa small molecule.

Embodiment 58

The method of any one of embodiments 35-57, wherein the composition isan aqueous solution.

Embodiment 59

The method of any one of embodiments 35-58, wherein the composition isat a pH from about 6.0 to about 7.5.

Embodiment 60

The method of embodiment 59, wherein the pH is about 6.8.

Embodiment 61

The method of any one of embodiments 35-60, wherein the compositionfurther comprises a buffering agent, solvent, reducing agent, a base, orcombinations thereof.

Embodiment 62

The method of any one of embodiments 35-60, further comprising sodiumphosphate, D2O, sodium azide, dimethyl sulfoxide, dithiothreitol orcombinations thereof.

Embodiment 63

The method of embodiment 62, wherein the sodium phosphate is present atabout 20 mM.

Embodiment 64

A method of identifying an SENP1 inhibitor, the method comprising:

combining an SENP1 polypeptide, a SUMO protein, and a test compound in areaction vessel;

allowing the SENP1 polypeptide, SUMO protein and test compound to form aSENP1-SUMO-compound complex; and

detecting the SENP1-SUMO-compound complex thereby identifying thecompound as a SENP1 inhibitor.

Embodiment 65

The method of embodiment 64, wherein one or more of the SENP1polypeptide, SUMO protein or test compound is labeled.

Embodiment 66

The method of embodiment 65, wherein the label is a fluorescent label.

Embodiment 67

The method of any one of embodiments 64-66, wherein the test compoundcomprises a fluorescent label.

Embodiment 68

The method of any one of embodiments 64-67, wherein binding is detectedby fluorescent polarization.

Embodiment 69

The method of embodiment 64, wherein binding is detected by detecting achange in the thermal properties of SENP1.

Embodiment 70

The method of embodiment 69, wherein the thermal property is the meltingtemperature of SENP1.

Embodiment 71

The method of any one of embodiments 64-70, wherein the SUMO is atruncated SUMO protein.

Embodiment 72

The method of any one of embodiments 64-70, wherein the SUMO comprisesamino acid residues 1-92 of the SUMO protein.

Embodiment 73

The method of any one of embodiments 64-70, wherein the SUMO proteincomprises SEQ ID NO:8.

Embodiment 74

The method of any one of embodiments 64-70, wherein the SUMO proteincomprises SEQ ID NO:9.

Embodiment 75

The method of any one of embodiments 64-74, wherein the SENP1polypeptide comprises SEQ ID NOs:1, 2, 3, 4, 5, 6, or 7.

Embodiment 76

The method of any one of embodiments 64-74, wherein the SENP1polypeptide comprises amino acid residue 603 of SEQ ID NO:1.

Embodiment 77

The method of any one of embodiments 64-74, wherein the SENP1polypeptide comprises a mutation at amino acid residue 603 of SEQ IDNO:1.

Embodiment 78

The method of embodiment 77, wherein the mutation is C603S.

Embodiment 79

The method of any one of embodiments 64-74, wherein the SENP1polypeptide comprises amino acid residues 440-455, 463-473, 493-515,529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 80

The method of any one of embodiments 64 or 71-79, wherein the detectingis performed using nuclear magnetic resonance.

Embodiment 81

The method of embodiment 80, wherein the detecting comprises producingan NMR spectra of the SENP1-SUMO-compound complex and identifying achange in the NMR spectra relative to the absence of the test compound.

Embodiment 82

The method of embodiment 81, wherein the change is a change in thechemical shift of an amino acid in an active site of the SENP1polypeptide.

Embodiment 83

The method of embodiment 82, wherein the active site is a catalyticallyactive site.

Embodiment 84

The method of embodiment 82, wherein the active site is a site thatbinds to the SUMO protein.

Embodiment 85

The method of embodiment 82, wherein the amino acid is an amino acid ofSEQ ID NOs:3, 4, 5, 6 OR 7.

Embodiment 86

The method of embodiment 82, wherein the amino acid is selected from thegroup consisting of D550, H533, C603, W465, W534, L466, G531, C535,M552, G554, E469 and Q596.

Embodiment 87

The method of embodiment 82, wherein the amino acid is S603.

Embodiment 88

The method of embodiment 82, wherein the amino acid is amino acidresidue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQID NO:1.

Embodiment 89

The method of any one of embodiments 64-88, wherein the test compound isa small molecule.

What is claimed:
 1. A method of screening for a binding compound ofsentrin/SUMO-specific protease 1 (SENP1) comprising contacting acomposition comprising an SENP1-small-ubiquitin-like modifier (SUMO)complex with a test compound and detecting whether the test compoundbinds SENP1 or a fragment thereof by nuclear magnetic resonance.
 2. Themethod of claim 1, wherein the detecting comprises determining achemical shift for an amino acid in an active site of SENP1.
 3. Themethod of claim 2, wherein the chemical shift in the presence of thecompound is changed relative to the corresponding chemical shift in theabsence of the compound.
 4. The method of claim 3, wherein the aminoacid is selected from the group consisting of an amino acid residuecorresponding to D550, H533, C603, W465, W534, L466, G531, C535, M552,G554, E469, and Q596 of SEQ ID NO:1.
 5. The method of claim 1, whereinSENP1 comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, or
 7. 6. The method ofclaim 1, wherein SENP1 comprises a mutation at an amino acid residuecorresponding to amino acid residue 603 of SEQ ID NO:1.
 7. The method ofclaim 1, wherein the detecting comprises producing an NMR spectra of aSENP1-SUMO-compound complex and identifying a change in the NMR spectrarelative to the absence of the compound.
 8. The method of claim 7,wherein the change is a change in the chemical shift of an amino acid ofSEQ ID NOs: 3, 4, 5, 6 or
 7. 9. The method of claim 7, wherein thechange is a change in the chemical shift of the amino acid correspondingto the amino acid C603 of SEQ ID NO:1.
 10. The method of claim 7,wherein the change is a change in the chemical shift of an amino acidresidue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQID NO:1.
 11. The method of claim 1, further comprising a biochemicalassay.
 12. The method of claim 11, wherein the biochemical assay is anenzyme kinetics assay, gel based assay, bioluminescent assay, orfluorescent assay.